CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design

We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 3' degenerate core. A longer 5' non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).     

CODEHOP-mediated PCR – A powerful technique for the identification and characterization of viral genomes

Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) PCR primers derived from amino acid sequence motifs which are highly conserved between members of a protein family have proven to be highly effective in the identification and characterization of distantly related family members. Here, the use of the CODEHOP strategy to identify novel viruses and obtain sequence information for phylogenetic characterization, gene structure determination and genome analysis is reviewed. While this review describes techniques for the identification of members of the herpesvirus family of DNA viruses, the same methodology and approach is applicable to other virus families.     

A novel herpesvirus in the sanctuary chimpanzees on Ngamba Island in Uganda

Background  Recent studies in non-human primates have led to the discovery of novel primate herpesviruses. In order to get more information on herpesvirus infections in apes, we studied wild born captive chimpanzees.
Methods  Chimpanzees of the Ngamba island sanctuary, Uganda, were analyzed with pan-herpes polymerase chain reaction (PCR) targeting the herpesvirus DNA polymerase gene and the glycoprotein B gene. The obtained sequences were connected by long-distance PCR, and analyzed phylogenetically.
Results  Twenty-one of 40 individuals were infected with members of the Gammaherpesvirinae , two of them with a novel member of this subfamily. Phylogenetically, the novel virus fell into a clade of primate rhadinoviruses and the Kaposi sarcoma herpesvirus (human herpesvirus 8), representing a third distinct rhadinovirus in chimpanzees.
Conclusion  Non-human primates harbor several herpesviruses many of which are still unknown. This has implications to management of primates in sanctuaries requiring continuous updates on the management protocols to deal with potential occupational pathogens.     

Homologous-Restraint Polymerase Chain Reaction: An Efficient and Rapid Protocol to Clone Multiple Homologous Genes

In this article, we present a novel protocol, called homologous-restraint polymerase chain reaction (HRPCR), for cloning multiple homologous genes. One of the homologous genes was cloned by consensus-degenerate hybrid oligonucleotide (CODEHOP) polymerase chain reaction (PCR) and sequenced. Primers of HRPCR were designed with 20 to 30 nt inverted to the known gene before the 5' end of the CODEHOP primers. The amplification of the known gene was restricted owing to the loop of the PCR product or the incorrect binding of the primers and the template. As a result, only unknown genes could be cloned. This protocol proved to be simple, rapid, and efficient. We applied this protocol to clone the multiple homologous genes of beta-1,4-N,6-O-diacetylmuramidase from the genomic DNA of Streptomyces griseus.     

Failure to detect simian immunodeficiency virus infection in a large Cameroonian cohort with high non-human primate exposure

Hunting and butchering of wildlife in Central Africa are known risk factors for a variety of human diseases, including HIV/AIDS. Due to the high incidence of human exposure to body fluids of non-human primates, the significant prevalence of simian immunodeficiency virus (SIV) in non-human primates, and hunting/butchering associated cross-species transmission of other retroviruses in Central Africa, it is possible that SIV is actively transmitted to humans from primate species other than mangabeys, chimpanzees, and/or gorillas. We evaluated SIV transmission to humans by screening 2,436 individuals that hunt and butcher non-human primates, a population in which simian foamy virus and simian T-lymphotropic virus were previously detected. We identified 23 individuals with high seroreactivity to SIV. Nucleic acid sequences of SIV genes could not be detected, suggesting that SIV infection in humans could occur at a lower frequency than infections with other retroviruses, including simian foamy virus and simian T-lymphotropic virus. Additional studies on human populations at risk for non-human primate zoonosis are necessary to determine whether these results are due to viral/host characteristics or are indicative of low SIV prevalence in primate species consumed as bushmeat as compared to other retroviruses in Cameroon.     

First case of ranavirus-associated mass mortality in a natural population of the Huanren frog (Rana huanrenensis) tadpoles in South Korea

Globally, ranavirus is often responsible for the mass mortality of a variety of captive and wild amphibians. In Asia, several mass mortality cases of captive amphibians by ranavirus are known, but one mass mortality case in the wild has been reported in a non-endemic larval bullfrog population in Japan. In order to verify factors involved in mass mortality of Rana huanrenensis tadpoles (> 200 tadpoles) in a mountain stream in South Korea, we investigated possible infections by ranavirus, chytrid fungus, and lethal bacteria by conducting PCR assays of pathogens with specific primers. We found that all R. huanrenensis tadpoles collected (two alive and ten carcasses) showed positive PCR results for two different ranavirus primer sets targeting partial genes of a major capsid protein (MCP). The identified MCP sequence was more closely related to Rana catesbeiana virus JP MCP, isolated from invasive bullfrog tadpoles in Japan. We could not detect any lethal bacteria or chytrid fungus in the specimens. Our finding is the first report in Asia that ranavirus is involved in the mass mortality of endemic wild amphibians.     

DEFOG: a practical scheme for deciphering families of genes

We developed a novel efficient scheme, DEFOG (for "deciphering families of genes"), for determining sequences of numerous genes from a family of interest. The scheme provides a powerful means to obtain a gene family composition in species for which high-throughput genomic sequencing data are not available. DEFOG uses two key procedures. The first is a novel algorithm for designing highly degenerate primers based on a set of known genes from the family of interest. These primers are used in PCR reactions to amplify the members of the gene family. The second combines oligofingerprinting of the cloned PCR products with clustering of the clones based on their fingerprints. By selecting members from each cluster, a low-redundancy clone subset is chosen for sequencing. We applied the scheme to the human olfactory receptor (OR) genes. OR genes constitute the largest gene superfamily in the human genome, as well as in the genomes of other vertebrate species. DEFOG almost tripled the size of the initial repertoire of human ORs in a single experiment, and only 7% of the PCR clones had to be sequenced. Extremely high degeneracies, reaching over a billion combinations of distinct PCR primer pairs, proved to be very effective and yielded only 0.4% nonspecific products.     

Group-Specific Multiplex PCR Detection Systems for the Identification of Flying Insect Prey

The applicability of species-specific primers to study feeding interactions is restricted to those ecosystems where the targeted prey species occur. Therefore, group-specific primer pairs, targeting higher taxonomic levels, are often desired to investigate interactions in a range of habitats that do not share the same species but the same groups of prey. Such primers are also valuable to study the diet of generalist predators when next generation sequencing approaches cannot be applied beneficially. Moreover, due to the large range of prey consumed by generalists, it is impossible to investigate the breadth of their diet with species-specific primers, even if multiplexing them. However, only few group-specific primers are available to date and important groups of prey such as flying insects have rarely been targeted. Our aim was to fill this gap and develop group-specific primers suitable to detect and identify the DNA of common taxa of flying insects. The primers were combined in two multiplex PCR systems, which allow a time- and cost-effective screening of samples for DNA of the dipteran subsection Calyptratae (including Anthomyiidae, Calliphoridae, Muscidae), other common dipteran families (Phoridae, Syrphidae, Bibionidae, Chironomidae, Sciaridae, Tipulidae), three orders of flying insects (Hymenoptera, Lepidoptera, Plecoptera) and coniferous aphids within the genus Cinara. The two PCR assays were highly specific and sensitive and their suitability to detect prey was confirmed by testing field-collected dietary samples from arthropods and vertebrates. The PCR assays presented here allow targeting prey at higher taxonomic levels such as family or order and therefore improve our ability to assess (trophic) interactions with flying insects in terrestrial and aquatic habitats.     

Isolation of distantly related members in a multigene family using the polymerase chain reaction technique.

We have designed a strategy to isolate and identify genes (cDNAs) coding for distantly-related members within a large multigene family. We have used limited protein sequence information data to delineate conserved regions where members of a supergene family are related. Comparison of the nucleotide sequences of such conserved areas defined consensus sequences that were used for the synthesis of deoxynucleotide primers. Two forward and two reverse primers were synthesized, and four separate pairs of primer combinations were used under low stringency in polymerase chain reactions (PCR) to generate amplified DNA products. The PCR products were directionally cloned into the phage vector M13mp18. Each of four libraries was screened with radiolabeled PCR product generated using a pair of primers different from those used to generate the library. Using this approach on the supergene family of ligand-gated ion channels, we were able to isolate and identify two novel subunits of neurotransmitter-operated ion channels.     

Overestimation of the abundance of sulfate-reducing bacteria in human feces by quantitative PCR targeting the Desulfovibrio 16S rRNA gene

The dominant genus of sulfate-reducing bacteria (SRB) in humans is Desulfovibrio, and quantitative PCR (QPCR) targeting the 16S rRNA gene is often used in assays. We show that the 16S rRNA gene assay overestimated SRB abundance in feces from 24 adults compared to QPCR assays using primers targeting two genes involved in SRB energy metabolism.     

The degenerate primer design problem

A PCR primer sequence is called degenerate if some of its positions have several possible bases. The degeneracy of the primer is the number of unique sequence combinations it contains. We study the problem of designing a pair of primers with prescribed degeneracy that match a maximum number of given input sequences. Such problems occur when studying a family of genes that is known only in part, or is known in a related species. We prove that various simplified versions of the problem are hard, show the polynomiality of some restricted cases, and develop approximation algorithms for one variant. Based on these algorithms, we implemented a program called HYDEN for designing highly-degenerate primers for a set of genomic sequences. We report on the success of the program in an experimental scheme for identifying all human olfactory receptor (OR) genes. In that project, HYDEN was used to design primers with degeneracies up to 10(10) that amplified with high specificity many novel genes of that family, tripling the number of OR genes known at the time.     

Comparative study of the transfection efficiency of commonly used viral vectors in rhesus monkey (Macaca mulatta) brains

Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system.In brain research,a series of powerful and novel gene editing technologies are based on these systems.Although many viral vectors are used in rodents,their full application has been limited in non-human primates.To identify viral vectors that can stably and effectively express exogenous genes within nonhuman primates,eleven commonly used recombinant adeno-associated viral and lentiviral vectors,each carrying a gene to express green or red fluorescence,were injected into the parietal cortex of four rhesus monkeys.The expression of fluorescent cells was used to quantify transfection efficiency.Histological results revealed that recombinant adeno-associated viral vectors,especially the serotype 2/9 coupled with the cytomegalovirus,human synapsin Ⅰ,or Ca2+/calmodulin-dependent protein kinase Ⅱ promoters,and lentiviral vector coupled with the human ubiquitin C promoter,induced higher expression of fluorescent cells,representing high transfection efficiency.This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates (NHPs).These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates.     

A method to type the potential angucycline producers in actinomycetes isolated from marine sponges

Angucyclines are aromatic polyketides with antimicrobial, antitumor, antiviral and enzyme inhibition activities. In this study, a new pair of degenerate primers targeting the cyclase genes that are involved in the aromatization of the first and/or second ring of angucycline, were designed and evaluated in a PCR protocol targeting the jadomycin cyclase gene of Streptomyces venezuelae ISP5230. The identity of the target amplicon was confirmed by sequencing. After validation, the primers were used to screen 49 actinomycete isolates from three different marine sponges to identify putative angucycline producers. Seven isolates were positively identified using this method. Sequence analysis of the positive amplicons confirmed their identity as putative angucycline cyclases with sequence highly similar to known angucycline cyclases. Phylogenetic analysis clustered these positives into the angucycline group of cyclases. Furthermore, amplifications of the seven isolates using ketosynthase-specific primers were positive, backing the results using the cyclase primers. Together these results provided strong support for the presence of angucycline biosynthetic genes in these isolates. The specific primer set targeting the cyclase can be used to identify putative angucycline producers among marine actinobacteria, and aid in the discovery of novel angucyclines.     

Duplication and expression analysis of multicopy miRNA gene family members in Arabidopsis and rice

To understand the expansion ofmulticopy microRNA （miRNA） families in plants, we localized the reported miRNA genes from Arabidopsis and rice to their chromosomes, respectively, and observed that 37% of 117 miRNA genes from Arabidopsis and 35% of 173 miRNA genes from rice were segmental duplications in the genome. In order to characterize whether the expression diversification has occurred among plant multicopy miRNA family members, we designed PCR primers targeting 48 predicted miRNA precursors from 10 families in Arabidopsis and rice. Results from RT-PCR data suggest that the transcribed precursors of members within the same miRNA family were present at different expression levels. In addition, although miRl60 and miR162 sequences were conserved in Arabidopsis and rice, we found that the expression patterns of these genes differed between the two species. These data suggested that expression diversification has occurred in multicopy miRNA families, increasing our understanding of the expression regulation of miRNAs in plants.     

Development of a real‐time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus

Aims: The goal of this study was to develop and to optimize molecular tools to detect the presence of Torque teno virus (TTV) in swine and cattle. A novel real‐time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect both genogroups of TTV strains. Methods and Results: Oligonucleotide primers and hybridization probes were designed based on sequence analysis of the noncoding region, a highly conserved part of the genome. The real‐time PCR assay specifically detected bovine and porcine TTV DNA without cross‐amplification of other common pathogens. The assay was compared with conventional PCR and nested‐PCR assays for the detection of porcine genogroups 1 and 2 and bovine TTV on plasma and faecal samples, and the assay was found faster, more reliable and reduced the risk of false positive results. Conclusions: The real‐time PCR assay provided better detection results for the two TTV genogroups in both swine and cattle compared to the conventional PCR assays. Significance and Impact of the Study: This new TaqMan PCR assay will be a useful tool for the detection of animal TTV strains, to evaluate the viral load from animal host and finally to identify the presence of these viruses in the agri‐food continuum.     

A sequence-independent strategy for detection and cloning of circular DNA virus genomes by using multiply primed rolling-circle amplification

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with phi29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 x 10(4)-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.     

Vaccine research efforts for filoviruses

Ebola and Marburg viruses belong to the family Filoviridae, and cause acute, frequently fatal, haemorrhagic fever in humans and non-human primates. No vaccines are available for human use. This review describes the status of research efforts to develop vaccines for these viruses and to identify the immune mechanisms of protection. The vaccine approaches discussed include DNA-based vaccines, and subunit vaccines vectored by adenovirus, alphavirus replicons, and vaccinia virus.