• Avian influenza virus (AIV) belongs to the influenza type
A genus of the
Orthomyxoviridae family of
RNA viruses. It is a highly mutable virus, with the
Haemagglutinin (HA) and the
Neuraminidase (NA) genes being the most variable. To date, 16 HA and 9 NA subtypes have been identified.
•C.W.Lee and Y.M. Saif. Avian influenza virus. Comparative
Immunology, Microbiology & Infectious Diseases, 32:301-310,
2009
• Rapid AIV subtype identification is critical for accurate diagnosis of human infections, effective response to epidemic outbreaks, and global-scale surveillance of highly pathogenic subtypes.
• Polymerase Chain Reaction (PCR) has become the method of choice for virus subtype identification due to its high sensitivity and specificity, fast response time, and affordable cost [Suarez et al. 2007].
• In [Duitama et al. 2009] we presented an open source software tool, called PrimerHunter, that can be used to select highly sensitive and specific primers for virus subtyping.
We have also confirmed the sensitivity and specificity of PrimerHunter's primers for hemagglutinin (HA) subtyping of Avian influenza virus using individual real time PCR (R-PCR) reactions testing the presence of each subtype.
• To increase sensitivity of detection when the quantity of viral
RNA is limited, we propose using PCR reactions with pools of subtype-specific primer pairs designed so that each subtype yields a unique amplification pattern.
• In this poster we describe an integer linear program (ILP) for designing the minimum number of uniquely decodable pools subject to primer non-dimerization constraints.
We also present results of real time RT-PCR reactions demonstrating the sensitivity of the assay for NA subtyping of AIV.
• Complete Avian Influenza
NA sequences from North
America available in NCBI flu database [Bao et al.
2008] as of March 2008
• Phylogenetic analysis detected a N1 sequence mislabeled as N4
• For each subtype N i
, we used all available N i sequences as targets, and all sequences with different subtype as non-targets
• PrimerHunter was run with default parameters: primer length 20 25, amplicon length 75 200 , GC content 25% -
75% , Max mononucleotide repeat 5 , matching mask M =
11, no required 3 ’ GC clamp, primer concentration 0.8
μM , salt concentration 50mM, T min_target
= T max_nontarget
= 40 o C
• Between 7 and 9,665 pairs of primers were found by
PrimerHunter for each subtype, full results are available at http://dna.engr.uconn.edu/software/PrimerHunter/
Subtype
N1
N2
N3
N4
N5
N6
N7
N8
N9
110
241
65
15
32
77
22
84
42
Targets
Non-
Targets
Avg. %
Dissimilarity
Forward
Primers
Reverse
Primers
578
447
623
673
656
611
666
604
646
8.8
11.7
8.8
7.1
6.4
10.3
7.2
6.7
6.6
97
77
45
370
355
29
97
140
292
71
44
61
360
353
43
103
211
305
Primer
Pairs
553
234
113
9665
8380
7
480
1785
6310
• To reduce the number of reactions needed to identify the subtype of a sample we perform RRT-PCR reactions with pools of primer pairs.
• Pools must be designed so that the result of these reactions uniquely identifies the subtype present in the sample. As noted by [Rash and Gusfield 2002], this is equivalent to ensuring that for every pair of subtypes there is a pool that results in a positive signal for one but not for the other.
• Additional constraints:
 Each subtype results in positive amplification for at least one pool
 Bounded pool size (no more than m pairs in a pool)
 No primer dimers (set of pairs of subtypes whose primers are predicted to form dimers is denoted by D ; we used the autodimer tool at the National Institute of Standards and Technology with a total score threshold of 4 to predict primer dimers)
• Optimization objective:
 Minimize the number of pools
 Subject to this, minimize total size of pools
• Notations
 N = number of subtypes ( N =9 for NA subtyping)
 P = set of of candidate pools, i.e., subsets of size at most m of {1, …,N} that do not contain any pair in D
 x p
= 0/1 variable set to 1 if pool otherwise p is selected and to 0
 M = constant larger than N |P|:
• We selected for each NA subtype one pair of primers
• For each pair, detection limits were determined using RRT-PCR on serially diluted RNA standards
• 10 potential primer dimers were identified using autodimer
Position of PCR products in the NA segment
• The ILP formulation had 85 variables (pool candidates) and 45 constraints that was solved to optimality in a fraction of a second using CPLEX; the four resulting pools are shown below
Primer Pool N1 N2 N3 N4 N5 N6 N7 N8 N9
A (2,6,7) + + + -
B (4,5,7,8) -
C (3,5,9) -
D (1,4,6,9) +
-
-
-
-
+
-
+
-
+
+
+
-
-
-
+
+
-
-
+
-
+
-
+
• Experiments show that RRT-PCR with primer pools can detect and differentiate NA RNA of all nine subtypes extracted from AIV-infected allantoid fluids.
• Amplification and dissociation properties of N4 RNA in primer pool tests are shown below; full results will be presented in [Huang et al 2010].
• In this study, 9 pairs of neuraminidase (NA) subtype-specific primers were designed and successfully used in real time
RT-PCR with four primer-pool reactions to differentiate 9 NA subtypes of AIV
• The RRT-PCR assays are sensitive and can detect in vitro transcribed RNA of different NA subtypes ranging from 176 to
4000 copies per reaction, or 2-30fg of AIV RNA
• The assays also possess good specificity. There was no cross reaction between RNA of different NA subtypes in RRT-
PCR with each subtype-specific primers, and no amplification was displayed for RNA of IBV, IBDV, NDV
• This study validated further the powerful function of Primer
Hunter for the design of subtyping primers and also introduced a sensitive and specific method for NA subtyping of AIV
• The quadruplicate primer pool test described decreases the amount of reactions needed to differentiate NA subtypes from
9 to 4. This reduces costs and labor, and could also improve sensitivity of detection when the quantity of viral RNA is limited
• Y. Bao, P. Bolotov, D. Dernovoy, B. Kiryutin, L. Zaslavsky, T. Tatusova,
J. Ostell, and D. Lipman. The influenza virus resource at the national center for biotechnology information. J. Virol., 82(2), pp. 596-601,
2008
• J. Duitama, D.M. Kumar, E. Hemphill, M. Kahn, I.I. Mandoiu, and C.E.
Nelson. PrimerHunter: A Primer Design Tool for PCR-Based Virus
Subtype Identification, Nucleic Acids Research 37, pp. 2483-2492,
2009
• Y. Huang, M. Kahn, and I.I. Mandoiu. Manuscript in preparation
• S. Rash and D. Gusfield. String barcoding: Uncovering optimal virus signatures, Proc. 6th Annual International Conference on
Computational Biology, pp. 254-261, 2002
• D. Suarez, A. Das, and E. Ellis. Review of rapid molecular diagnostic tools for avian influenza. Avian Diseases, 51, pp. 201-208, 2007