Universal Primer for Early and Rapid Detection of Nucleopolyhedroviruses of Multiple Species Using Polymerase Chain Reaction

A technique using the polymerase chain reaction (PCR) was developed for detection of the nucleopolyhedrovirus (NPV) polyhedrin gene. 152 nucleotide sequences of polyhedrin gene were compared in pairwise and multiple alignment sequences. Eleven highly conserved DNA sequences within the coding region of the polyhedrin gene were identified. Two candidate regions were targeted for amplification and consequently one pair of degenerate PCR primers was designed to produce fragments of about 355 bp. The NPVs tested by this technique were Autographa californica (AcMNPV), Bombyx mori NPV (BmNPV), Hyphantria cunea NPV (HcNPV), Lymantria dispar NPV (LdNPV), Spodoptera exigua NPV (SeNPV), S. litura NPV (SlNPV), Spodoptera littoralis NPV (SpliNPV) and nine local NPV isolates. Furthermore, three randomly chosen PCR products were cloned and sequenced. The sequencing data showed that the three PCR products were fragments of polyhedrin gene. Conclusively, this technique would be useful in monitoring the environmental fate, distribution of NPVs, release of the wild type and recombinant NPVs and quality control studies of baculoviral insecticides as well.


INTRODUCTIION
Baculoviruses have a large circular double-stranded DNA genome ranging from approximately 80 to 180 kb in size (Blissard and Rohrmann, 1990).They are considered to be the largest and most broadly studied insect viruses because they are of great interest and utility to large cross-sections of agricultural and biomedical research community.They have been thoroughly investigated due to its potential as insect control agent (Wood and Robert, 1991), and as a vector expressing various heterologous genes (Summers andSmith, 1987, Choi et al., 1999).Although Murphy et al. (1995) have reported baculovirus infections in over 600 insect species in the order of Lepidoptera, Hymenoptera, Diptera, Coleoptera, Neuroptera, Trichopera and Thysanura, as well as in the Crustaceae order Decapoda (shrimps), it is recently confirmed that only those derived from orders Lepidoptera, Hymenoptera, and Diptera are members of the family Baculoviridae (ICTV, 2009).Those from Orthoptera were classified as pox viruses, isolates from Coleoptera are so far not assigned (ICTV, 2009).Neuropterans were infected under laboratory conditions and larval death from the virus infection was not documented.Shrimp viruses have now been classified as Nimaviridae in the genus Whispovirus and are no longer baculoviruses (Marks et al., 2005).The commercialization and release of recombinant viruses in the environment created the concern that they might cause ecological disturbances, such as displacement of native microorganisms, adverse effects on non-target organisms and the horizontal transfer of DNA into non-target organisms (Leung et al., 1994).For the above mentioned reasons, many authors were interested in developing an accurate and easy diagnostic method for early and rapid detection of NPV infections (Wang et al., 2000, Christian et al., 2001, Moraes and Maruniak, 2001, Woo, 2001, Lange et al., 2004, Jehle et al., 2006, Murillo et al., 2006).
Polyhedrin is the major component of polyhedra and has often been studied.After the first report about localization of the polyhedrin gene in AcNPV (Vlak and Smith, 1982), Iddekinge et al. (1983) determined its nucleotide sequences.At the last count, there was published data on the complete genome sequence of some 41 or more NPVs.Polyhedrin is a protein of about 245 to 250 amino acids, and appears to be the most highly conserved NPV protein.These characteristics lead to the use of polyhedrin sequences as the base of NPV phylogenetic studies (Zanotto et al., 1993).
The polymerase chain reaction (PCR) is a highly sensitive technique, which amplifies target DNA sequences and does not employ radioactive material.PCR has been extensively used to detect many organisms such as animal, human, plant, and various pathogens.Webb et al. (1991) reported the use of PCR to screen baculovirus expression vector recombinants in cell cultures.Burand et al. (1992)  The aim of the present study was to design degenerate primer set to detect multiple NPVs using PCR technique.The ability to detect NPV polyhedrin will be a useful tool in studies seeking to rapidly elucidate a polyhedrin gene structure, to monitor the release of the wild type as well as genetically engineered NPVs, and to isolate NPVs in the natural environment.

Polyhedrin sequence data
All polyhedrin sequences of nucleopolyhedrovirus (NPV) available in March 2010 from GenBank, EMBL, and DDBJ were downloaded.152 nucleotide sequences were aligned using ClustalX software.Neighbour joining tree was examined using ClustalX and no divergent sequences were identified.In addition, the alignment was manually corrected by shifting sequences in places, for some sequences possessed large spans of unique deletions or insertions which threw off the alignment algorithm.

Selection of highly conserved genome regions for primer design
The term "conserved genomic regions" used here is defined as genome regions that have most frequently presented nucleotide sequences.To identify the highly conserved regions eligible for primer design, pairwise scan for the sequences was done and base by base alignment output file was produced using Mega4 and/ or ClustalX softwares.The most frequently presented base in the same coordinate for all sequences of the alignment was detected.The output (FASTA file) was then analyzed by ClustalX software to select candidate conserved regions for primer design.A candidate region was defined as a site within the polyhedrin open reading frame (orf) that had 17+ bases from the 3' end and with a base frequency of 0.80+.Candidate conserved regions were identified by calculating redundancy scores and the average dominant base counts.Average dominant base counts were calculated by summing the number of occurrences of the most common base at each position in a window length of 20 bases and averaging those counts across all positions in the window.

Primer design
The distance between conserved regions was taken into account when selecting conserved sites as was the potential for using mixed bases or deoxyinosines, to enhance bonding at variable positions.Standard nucleotides were preferred close to the 3' termini of the oligonucleotides.The different parameters of primer design (length and sequence, GC content, Tm,........etc.) were taken into consideration.One set of degenerate primers common for the whole group was designed.Primers were designed to amplify 355 bp within the polyhedrin gene.
The accession numbers of polyhedrin nucleotide sequences used in this study are listed in Table (1).

Viruses, cell lines and insects
The nucleopolyheroviruses tested in this study were as follows: Autographa californica (AcMNPV), Bombyx mori NPV (BmNPV), Hyphantria cunea NPV (HcNPV), Lymantria dispar NPV (LdNPV), Spodoptera exigua NPV (SeNPV), S. litura NPV (SlNPV), Spodoptera littoralis NPV (SpliNPV) and nine local NPV isolates.The AcMNPV, BmNPV and HcNPV were propagated in Sf9 cells maintained at 27°C in a TC-100 medium (Gibco-BRL, USA) that was supplemented with 10% fetal bovine serum (Gibco-BRL, USA).The SeNPV, SlNPV, SpliNPV, LdNPV and local isolates were propagated in S. littoralis and L. dispar larvae.Routine cell culture maintenance and virus production procedures were carried out according to O' Reilly et al. (1992).Insect colonization and maintenance of the cotton leafworm, Spodoptera littoralis, was done in the insectary of Department of Entomology, Faculty of Science, Cairo University under highly controlled conditions from 1995 to date.The colony was maintained in the laboratory according to Seufi (2008).These insects were used for viral propagation and purification assays.

Virus DNA purification
Virus DNA was extracted from purified, semipurified polyhedra collected from infected cells and insect larvae.Total genomic DNA was also extracted from insect larvae.The virus isolates were successfully propagated and purified following the method described by Lacey et al. (2002).To extract virus DNA, purified or semipurified polyhedra were resuspended in a 0.1 M sodium carbonate solution (0.1 M Na2CO3, 0.17 M NaCl, 0.01 M EDTA, pH 10.9), and incubated at 37°C overnight with a final concentration of 0.5 mg/ ml of proteinase K (Sigma, USA) and 1% of SDS.A further extraction with phenol Fatma H. Galal 60 and chloroform: isoamylalcohol (24:1) was performed and the DNA was ethanol-precipitated.The DNA was resuspended in a TE buffer (10 mM Tris-HCl, 1mM EDTA, pH 8.0).

PCR amplification
PCR amplification was performed according to Saiki et al. (1988) with minor modifications.Total DNA was extracted from the NPV isolates and the DNA segment was amplified using two primers designed based on conserved nucleotide sequences of 152 different polyhedrin sequences.Sequence of the forward and reverse primers used in this study, their length, GC content and base counts were shown in Table (3 and 4).Total reaction volume was 50 μl which contained 1× PCR buffer (Promega), 1.5 mM MgCl2, 200 μM dNTPs, 2.5 U Taq DNA polymerase (Promega), 100 ng of each primer and 30 ng of template DNA.The amplification program used was 3 min at 94°C (hot start), 1 min at 94°C, 2 min at 55°C and 2 min at 72°C for 35 cycles followed by one cycle of 72°C for 7 min.PCR amplification was carried out in a DNA thermal cycler (Model 380 A, Applied Biosystems, CA, USA).

Selection of Candidate Conserved Region for Primer Design
Alignment of the 152 NPV polyhedrin sequences were used as a guide to enable identification of conserved sequences of the gene to be used in the design of degenerate oligonucleotide primers for PCR.No potentially useful conserved sites were identified in the first complete multiple alignment, utilizing all available sequences in GenBank, EMBL, and DDBJ databases.However, once divergent sequences were removed, eleven conserved regions were identified.Two candidate regions (from 154 to 172 and 465 to 505 relative to the Cotesia marginiventris NPV polyhedrin gene (Acc# EF418027)) with relatively low levels of degeneracy were selected to design primers (Table 2).One set of degenerate PCR primers (Polh152F and Polh152R) was designed from these regions.

Primer Selection
The Polh152F and Polh152R primers were designed to amplify 355 bp within the polyhedrin gene (based on 152 polyhedrin sequences).The sequences of The Polh152F and Polh152R primers and the base count of the respective viral DNAs are shown in Tables (3 and 4).Degenerate sites were considered when low base by base frequency was produced by multiple alignment in the candidate regions.The primers were selected on the basis of having relatively low levels of degeneracy.The bases represented in lower than 5% in the base count were not considered in designing the primers (Tables 3 and 4).To compensate for the primer multiplicity, a slightly higher primer concentration (50 pmole per 50 μl reaction) was used in the PCR.

Experimental verification
Two degenerate primers were designed to anneal within the orf of polyhedrin gene (Polh152F and Polh152R).The degenerate PCR primer set successfully amplified the expected polyhedrin DNA fragment (355 bp) from the AcMNPV, BmNPV, HcNPV, LdNPV, SeNPV, SlNPV, SpliNPV as well as from nine local NPV isolates.

Universal Primer for Early and Rapid Detection of Nucleopolyhedroviruses of Multiple Species 61
Table 3: Sequence and base counts of the forward primer based on the first candidate conserved region of 152 polyhedrin sequences.Base count less than 5% was neglected in primer design.

Base count
Forward primer (5`--- V= G, A or C, D= A, T or G, B= C, G or T, R= A or G, Y= C or T.
Table 4: Sequence and base counts of the reverse primer based on the eighth candidate conserved region of 152 polyhedrin sequences.Base count less than 5% was neglected in primer design.

Base count
Reverse primer (5`---3`) Non-specific amplification products were not observed for tested NPVs.In total all of the nine NPV species tested were amplified, seven species and two local isolates have been tested with these primers (Fig. 1).Three randomly chosen PCR products were cloned into pGEM-T vector and sequenced.The sequencing results showed that the three PCR products were fragments of polyhedrin gene (data not shown).

DISCUSSION AND CONCLUSION
Polyhedrin gene of NPVs encodes for the matrix protein of the virus occlusion body and is one of the most conserved baculovirus genes (Jehle, 2004).This gene was proved to be the most suitable gene in baculoviruses for developing generic amplification technique (Woo, 2001).Seufi (2008) characterized a highly conserved polyhedrin region of 405 bp molecular size.He reported that alignment results of this conserved region with the other published sequences produced significant alignment with 111 baculovirus isolates.The percentage of homology ranged between 99% for SpliNPV (Acc# D01017) and 78% for Plusia orichalcea NPV (Acc# AF019882).At the level of amino acid sequence, the percentage homology of ranged between 100% for S. littoralis polyhedrins (Acc# AAC33752 and AAR04375) and 81% for Attacus ricini polyhedrin (Acc# P31036).In addition, lepidopteran polyhedrin genes show about a 50% amino acid identity with granulovirus granulins, and a 40% identity with hymenopteran NPV polyhedrin (Rohrmann, 1992).These high similarities could enhance the strategy to design universal primers for rapid and early detection of NPV-infection.The advantage of such approach is that it utilized all NPV polyhedrin sequences available in the international databases (152 sequences) and simple public software programs to select optimal candidate regions in genomic sequences Fatma H. Galal 62 for amplification.Such approach is unlikely to produce significant bias towards any one species, especially when there is no bias in the multiple sequence alignment which the approach was based on.These primers make it possible to efficiently amplify DNA from many NPV species and allow further search for unknown NPV species.
Many published reports that investigated polyhedrin gene depended primarily on a Southern hybridization using probes of the polyhedrin gene of other previously identified viruses.However, this technique is efficient only if the similarity between polyhedrin genes of the target NPV and probe NPV is high.Therefore, many limitations will arise when the study based mainly on Southern technique.One major limitation is that this technique requires multiple probes of various NPVs for detection of baculovirus.Also, traditional serological methods based on neutralization and fixed cell ELISA have proven effective for identifying baculoviruses (Brown et al., 1982).However, difficulties in interpreting antigenic cross reactivity or failure to identify relatively close antigenic relationships were common complains in this technology.Moreover, serology is time consuming, requires highly experienced personnel and is less precise than nucleotide sequence determination.
Generally, the use of PCR technology for virus detection, identification and characterization is a basic tool in many virological laboratories (Moraes and Maruniak, 1997, Moraes et al., 1999, Murillo et al., 2006).Indeed, a good set of primers for nucleopolyhedrovirus detection is a powerful tool for large nucleopolyhedrovirus sample screening.PCR technique is preferred because it is easy, fast, sensitive and reliable.In addition, it does not utilize radioactive materials.Although attempts to detect nucleopolyhedroviruses from soil and insects have been made using PCR techniques, it was limited to narrow NPV species (Webb et al., 1991, Moraes and Maruniak, 1997, Moraes et al., 1999).Woo (2001) designed a pair of degenerate primers (based on conserved amino acid sequence of 26 different NPVs) to detect multiple NPVs using PCR.One major problem with degenerate primers is that the concentration of some permutations in the mixture is so small that amplification is effectively inhibited (due to their great multiplicity).It was believed that the redundancy of Polh152F and Polh152R was insufficient to cause this problem.The capacity of Polh152F and Polh152R primers potentially to amplify all NPVs made them an invaluable diagnostic and taxonomic tool for virology.The ability of these primers to amplify DNA from local isolates of NPV may demonstrate their capacity to define novel NPV species.
In conclusion, the PCR primer set employed in this study was chosen from highly conserved sequences within the polyhedrin-coding region.Therefore, the possibility of amplification of multiple nucleopolyhedroviruses was more enhanced.The present study introduced a highly sensitive method for multiple nucleopolyhedrovirus detection.Higher sensitivity and cost-efficiency enabled the researcher to identify the structure of the polyhedrin gene rapidly.The amplification of highly specific and abundant products obtained in this study suggests that this method might be useful to detect nucleopolyhedroviruses with low amounts of DNA in the environment.Conclusively, the method described in this paper is universal, powerful, and could be used in the future to study the environmental fate of wild type or genetically modified recombinant NPVs.It may be usefull in quality control studies of baculoviral insecticides as well.

Table 1 :
Nucleopolyhedroviruses and sources of sequence information for sequences used in the present study.

Table 2 :
Number, length and location of the identified