Avian influenza (AI) is a significant disease of many bird species, and some subtypes represent a threat to humans. Low pathogenic (LP) avian influenza viruses (AIV) are known to circulate in wild birds, and recently the Asian H5N1 highly pathogenic (HP) AIVs have also been found in wild birds (1,2). This wild bird reservoir presents an ongoing threat of AIV introduction into domestic poultry flocks. As a result of the recent emergence of Asian H5N1 viruses that are capable of zoonotic spread, wild and domestic bird surveillance for AIV has increased worldwide, requiring the development of rapid and precise methods to characterize the isolates (3). The high capacity for mutation in RNA viruses (genetic drift), and the generation of reassortant viruses (genetic shift) in wild birds make it impossible to predict the genetic composition of new isolates. Also, due to the relatively few nucleotide differences within the hemagglutinin gene between LP viruses and the highly pathogenic phenotype, LP viruses have the capacity to mutate to HP (4). Current efforts to characterize new isolates have focused on the development of DNA microarrays, real-time PCR, or rapid sequencing of real-time PCR products (5–11). The common pitfall for each of these methods is that detection relies upon primers that are designed from existing sequences, which presumes that the unknown virus will resemble previously sequenced viruses. In addition, with the exception of complete genomic sequencing, most of these methods characterize small genomic regions, thus offering a partial view of the virus. Another issue that affects the rapid and precise AIV characterization is the frequent occurrence of mixed infections in wild birds with viruses of different serotypes. In these cases, any primer-specific method will amplify the viral product most homologous to the primer(s) of choice, but not necessarily the most abundant or representative of the viral population infecting the birds.
Random genome sequencing represents an unbiased and thorough alternative that has been widely used to characterize the genomes of large DNA viruses (12), but RNA viruses with fragmented genomes have been resilient to this approach. Currently, 100% of the genomic characterization of AIV carried out by The Institute for Genome Research (TIGR) and the NIH is based upon primer-specific approaches (msc.tigr.org/influenza/ index.shtml) that require previous antibody-based typing of the hemagglutinin and neuraminidase genes for primer selection. Despite serotyping, viruses with genomes that are highly divergent from the primer’s sequences often cannot be readily sequenced. To overcome these limitations, we have designed and optimized a protocol that follows two basic principles: (i) random amplification of total RNA, and (ii) random selection of colonies followed by sequencing and assembly.
The A/Quail/PA/20304/98 (H7N2) LPAI virus was obtained from the Southeast Poultry Research Laboratory (SEPRL) repository and grown in 11-day-old embryonating chicken eggs. RNA was extracted from allantoic fluid using TRIzol® LS (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions; 750 μL TRIzol LS reagent were added to 250 µL allantoic fluid, vortex mixed, and incubated at room temperature for 7 min. RNA was separated into the aqueous phase with the addition of 200 μL chloroform and precipitated with isopropanol. After one wash with 70% ethanol, RNA was dried and resuspended in RNase-free water. Amplification of total RNA was performed with the TransPlex™ whole transcriptome amplification (WTA) kit (Rubicon Genomics, Ann Arbor, MI, USA) using conditions for library synthesis and amplification (program WTA1). A library synthesis step with primers comprised quasi random 3′ ends and universal 5′ ends was performed, and universal PCR primers were used to amplify the library (patent no. WO/2004/081225). Amplified products were separated on a 1% agarose gel; fragments ranging from 300–3000 bp were excised and eluted using the QuickClean 5M gel extraction kit (GenScript, Piscataway, NJ, USA), and the samples quantified using a standard spectrophotomer. These segments were cloned using pCR®2.1 TOPO® (Invitrogen), and all white colonies were sequenced.
To demonstrate the practical and rapid characterization of AIV, white bacterial colonies were randomly selected for sequencing. Sequencing reactions were performed with fluorescent dideoxynucleotide terminators in an ABI 3700 automated sequencer (Applied Biosystems, Foster City, CA, USA). Nucleotide sequence editing and analysis were conducted with Codon Code Aligner 1.6.3 (CodonCode, Dedham, MA, USA). Assembled contigs were compared with the GenBank® database by Basic Local Alignment Search Tool (BLASTn) analysis. For A/Quail/PA/20304/98 and A/Quail/PA/20304/98-P15, sequencing results produced consensus sequences totaling 11,013 and 12,186 bases, which correspond to 85% and 93.2% coverage of the entire genome, respectively. Of the 617 and 634 reads analyzed, 75.68% and 64.35%, respectively, corresponded to AIV genomic sequences, while chicken and bacterial contaminant ranged from 1.7% to 2.3%, and the remainder corresponded to either bad quality sequences or sequence that did not assemble into contigs. The capacity of AIV to grow at high titers in allantoic fluids was sufficient to avoid viral purification and to prevent contamination with host nucleic acids. The diffuse smear in Figure 1A illustrates the randomness of the amplification procedure and the range of sizes of cDNAs obtained. The broad genome coverage achieved for most AIV genomic fragments is shown in Figure 1B, which allows for clear identification of the virus serotype utilizing BLAST analysis (data not shown).
In summary, the use of nonpurified AIVs obtained from allantoic fluids, combined with highly efficient cDNA generation and amplification provided a highly random and complete representation of the AI viral genes in the cloned products. Sequence comparison of two complete gene fragments (HA and NS1/NS2) revealed >99% identity (980 identities over 981 nucleotides and 847 identities over 848 nucleotides, respectively) to the previously reported sequences (GenBank accession nos. AY241656.1 and AY240924.1). Because this approach does not rely upon the use of specific PCR primers, it has the potential to detect quasispecies, mixed infections, and the presence of other viral and nonviral contaminants in the inoculum, which in turn, allows for a better interpretation of the pathogenicity tests. In our experience, results from the intracerebral pathogenicity index (ICPI) or mean death time (MDT) assays are often inaccurate due to the presence of mixed viral infections or other undetected pathogens. This method, although not intended to replace the use of sequence specific primer amplification, provides a relatively rapid sequencing alternative (48 h from RNA isolation to virus identification) that may also be applicable to other RNA viruses.
ACKNOWLEDGMENTS
The author gratefully acknowledges Dr. David E. Swayne for the virus, Joan Beck, Kristin Zaffuto and Dawn Williams-Coplin for technical assistance, Dr. David Suarez, Laszlo Zsak, Samadhan Jadhao, Michael Day, and L.M. Kim for critical comments, and the South Atlantic Area Sequencing Facility for nucleotide sequencing. This work was funded by USDA CRIS project no. 6612-32000-048. Mention of trade names or commercial products in this manuscript is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
COMPETING INTERESTS STATEMENT
The author declares no competing interests
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This article was originally published at the BioTechniques 43:188-192 Vol.43, No.2 pp. 188-192, 2007.
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