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Development of Rabbitpox Virus and Cowpox Virus Real-Time PCR Assays

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Development of Rabbitpox Virus and Cowpox Virus Real-Time PCR Assays 030 (A) Development of Rabbitpox Virus and Cowpox Virus Real-Time PCR Assays Jessica Shifflett, Sujatha Radhakrishnan, Kurt Langenbach BEI Resources/American Type Culture Collection Abstract Results Background Table 1: Specificity testing panel Figure 2: Amplification charts demonstrating specificity of the assays against the 21 Orthopoxviruses and 5 host cell lines Vaccinia virus is a prevalent tool in research due to its ease of manipulation into viral vectors or recombinant strains. The virus is particularly available at ATCC® (see Table 1). A. Rabbitpox virus qPCR assay with ATCC® VR-1591™. B. Cowpox virus qPCR assay with advantageous because it can be handled at BSL-2, occupies a wide host range and is inexpensive to propagate. The 41 recognized strains of NR-2641. Vaccinia virus have minimal variation at the genomic level and cross contamination of viral stocks is a potential problem. BEI Resources was BEI Resources/ATCC® A B tasked with the development of Rabbitpox virus and Cowpox virus real-time PCR assays in order to distinguish the viruses from other common laboratory strains of Orthopoxviruses. Catalog # Virus Species/Cell Line Strain Methods NR-2634 Vaccinia MVA The Rabbitpox virus assay was designed based on a whole genome multiple alignment of near neighbors. The Cowpox virus primers designed by S.N. Shchelkunov, et al (2005) were adapted for our use in real-time PCR by the addition of a newly designed specific probe. The NR-2635 Vaccinia Elstree specificities of the primer and probe sequences were verified using NCBI BLAST and tested against the 21 available Orthopoxviruses and 5 NR-2636 Vaccinia IHD host cell lines at ATCC®. Positive control plasmids for each assay were designed by inverting every other 10 nucleotides within the target NR-2637 Vaccinia Lederle Chorioallantoic region, excluding the primer and probe binding regions. This resulted in a positive control target with the same GC content and melting temperature as the “wild type” target. NR-2638 Vaccinia NYCBOH Introduction NR-2639 Vaccinia Western Reserve (TC adapted) Results NR-2640 Vaccinia Western Reserve (mouse adapted) The Rabbitpox virus and Cowpox virus assays are specific to the detection of Rabbitpox or Cowpox viruses respectively by both the NCBI NR-2641 Cowpox Brighton Red database and wet-lab testing against the more common laboratory strains. The assays reliably detect down to 100 molecule copies. Additionally, a spike-in study demonstrated that 10 copies of Rabbitpox virus could be detected and discriminated from neat Vaccinia virus NR-3021 Monkeypox Zaire 79 (CDC) strain MVA or Monkeypox virus strain 7-61 WRAIR. NR-3071 Monkeypox 7-61 WRAIR Conclusion ATCC® VR-838™ Raccoonpox Herman ATCC® VR-111™ Canarypox Wheatley c93 The popularity of Vaccinia virus strains in research requires a sensitive method to detect near-neighbor contaminants to insure the integrity of Figure 3: Amplification chart showing the Rabbitpox virus qPCR assay detecting 10 copies of the Rabbitpox positive control plasmid spiked experiments. Here, we have shown the development of a novel Rabbitpox virus real-time PCR assay and the adaptation of a Cowpox virus ATCC® VR-1591™ Rabbitpox Utrecht into neat Vaccinia virus MVA or Monkeypox virus 7-61 WRAIR (green). The Rabbitpox plasmid alone is shown in red (10 copies). Neat real-time PCR assay. ATCC® VR-363™ Swinepox Kasza Vaccinia virus and Monkeypox virus alone are shown in blue. NR-4928 Monkeypox US2003 NR-523 Monkeypox Zaire 79 (contaminated with CPX) NR-679 Monkeypox Zaire 79 (contaminated with CPX) Introduction NR-2323 Monkeypox Zaire 79 NR-2324 Monkeypox Zaire 79 (V79-1-005) The murky history of Vaccinia virus began when it was recognized that the original smallpox vaccine strain, Cowpox virus, was replaced ATCC® VR-937™ Tanapox Davis somewhere in its course of use with Vaccinia virus. Similar clinical manifestations make distinctions between the infection with the two viruses ATCC® VR-634™ Pseudocowpoxvirus TJS difficult and it is unknown when the switch from Cowpox to Vaccinia virus in the smallpox vaccination occurred1. Vaccinia virus continues to be used as a vaccine today, and has evolved through different attenuated, temperature sensitive and recombinant forms to prime the immune ATCC® CCL-81™ Vero 3 system against not only Variola virus, but also against a myriad of other pox viruses . The obscure origins of the virus coupled with its ATCC® CRL-1586™ Vero E6 continued importance in gene delivery and vaccine research underscore the requirement for a sensitive and specific identification method in experimental integrity. ATCC® CCL-7.1™ LLC-MK2 Derivative ATCC® CRL-2378.1™ MA-104 Clone 1 The ~190 kb Vaccinia virus genome is highly conserved not only among the 41 recognized strains of Vaccinia virus, but also among the other members of the Poxviridae family2,5. Over 200 genes are encoded in the viral genome, with 34 replication and assembly associated genes ATCC® CCL-171™ MRC-5 and an additional 52 accessory genes conserved between the Poxviridae family7. The challenge to find an oligonucleotide unique to a single strain within the species is great considering the high degree of homology. Figure 1: Amplification charts demonstrating sensitivity. A. The Rabbitpox virus assay reliably detects down to 100 copies with a PCR efficiency of 98.2%, R2 value of 0.998 and slope of -3.356. B. The Cowpox virus assay reliably detects a lower limit of 100 molecule copies with a PCR efficiency of 115.8%, R2 value of 0.968 and slope of -2.993. A Materials and Methods Conclusions The popularity of Vaccinia virus strains in research requires a sensitive method to detect near-neighbor contaminants to insure the integrity The real time quantitative polymerase chain reaction (qPCR) assay specific for Rabbitpox virus4 (AY484669.1) was designed based on a whole of experiments, especially in labs that manipulate related viruses. Here we have shown the development of a novel Rabbitpox virus qPCR genome multiple alignment of near neighbors including the following: Vaccinia virus Lister (AY678276.1), Vaccinia virus Western Reserve assay and the adaptation of a Cowpox virus qPCR assay that have already proved useful within the BEIR Virology Collection. The Cowpox (AY243312.1), Vaccinia virus Copenhagen (M35027.1), Vaccinia virus Ankara (AM501482), Cowpox virus Brighton Red (AF482758), Cowpox virus qPCR assay was used to identify suspected Cowpox contamination within two Monkeypox virus preparations (NR-523 and NR-679) virus GRI-90 (X94355.2), Monkeypox virus Zaire 1979-005 (DQ011155.1) and Camelpox virus CMS (AY009089). The clustal W alignment was and the Rabbitpox virus qPCR assay was shown capable of detecting Rabbitpox amidst Vaccinia virus and Monkeypox virus (Figure 3). done using Mobyle software at http://mobyle.pasteur.fr/cgi-bin/portal.py. The parameters were as follows: word size ktuple = 2, diagonals = 5, window size = 5, gap penalty = 10, gap extension penalty = 0.2, gap separation penalty range = 8, delay divergent sequence = 30. The selected forward and reverse primers amplify a range of Orthopoxviruses targeting the inverted terminal repeat region. Specificity is narrowed to Rabbitpox virus with the probe. References & Acknowledgements 1. Baxby, Derrick (1977) “The Origins of Vaccinia Virus” The Journal of Infectious Diseases 136(3):453-455. The Cowpox virus qPCR assay primers were designed by S.N. Shchelkunov et al (2005) within the B9R gene6. The target sequence was 102 aligned with near neighbors Camelpox virus M-96 (AF438165.1), Camelpox virus CMS (AY009089) and Taterapox virus Dahomey 1968 2. Gubser, C., S. Hué, P Kellam and G.L. Smith (2004) “Poxvirus genomes: a phylogenetic analysis” Journal of General Virology 85:105-117. (DQ437594) with NCBI Specialized BLAST Align at http://blast.ncbi.nlm.nih.gov/ to determine a Cowpox virus specific probe. doi:10.1099/vir.0.19565-0. The specificities of the primer and probe sequences were verified using NCBI BLAST and NCBI Specialized Primer-BLAST using the default B 3. Jacobs, B.L., J.O. Langland, K.V. Kibler, K.L. Denzler, S.D. White, S.A. Holechek, S. Wong, T. Huynh and C.R. Baskin (2009) settings. Additionally, the specificity of the Rabbitpox virus and Cowpox virus qPCR assays was tested against the available Orthopoxviruses “Vaccinia Virus Vaccines: Past, Present and Future” Antiviral Research 84(1):1-13. doi:10.1016/j.antiviral.2009.06.006. and common host cell lines at ATCC® (Table 1 and Figure 2). The 50 µL reaction volumes are composed of 5.0 µL DNA template, 0.25 µL iTaq™ DNA Polymerase (Bio-Rad® 170-8870), 5.0 µL 10x iTaq™ Buffer, 2.5 µL 50 mM MgCl , 1.0 µL 10 mM dNTPs (Bio-Rad® 170-8874), 1.5 2 4. Li, G., N. Chen, R.L. Roper, Z. Feng, A. Hunter, M. Danilla, E.J. Lefkowitz, R.M.L. Buller and C. Upton (2005) “Complete coding sequences µL10 µM forward primer (Rabbitpox BEIR NR-26769 and Cowpox BEIR NR-26772), 1.5 µL 10 µM reverse primer (Rabbitpox BEIR NR-26770 of the rabbitpox virus genome” Journal of General Virology 86:2969-2977. doi:10.1099/vir.0.81331-0. and Cowpox BEIR NR-26773), 0.5 µL 5 µM probe (Rabbitpox BEIR NR-26771 and Cowpox BEIR NR-26774) and 32.75 µL molecular biology grade water. The cycling conditions are a 2 minute 95°C hot start, followed by 40 cycles (Rabbitpox) or 45 cycles (Cowpox) of denaturing at 5. McLysaght, A., P.F. Baldi and B.S. Gaut (2003) “Extensive gene gain associated with adaptive evolution of poxviruses” Proceedings of the 95°C for 30 seconds, annealing at 62°C for 30 seconds (Rabbitpox) or 56°C for 45 seconds (Cowpox), and extending at 72°C for 45 seconds National Academy of Sciences 100(26):15655-15660.
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