The polymerase chain reaction (PCR) is an extremely sensitive assay that has many uses in retroviral-mediated gene transfer protocols. Because the majority of retroviral vectors used in current gene transfer protocols are based on the Moloney-murine leukemia virus (MMLV), we have designed primers which amplify a region of the psi packaging sequence from all MMLV retroviruses tested. This assay detects gene transfer by all MMLV-based vectors and is especially useful for the laboratory that routinely screens a number of different retroviruses for their gene transfer efficiency. Furthermore, we present here a novel technique for harvesting single colonies derived from hematopoietic stem/progenitor cells growing in methylcellulose medium that expedites and substantially improves the resulting quantitative estimates of retroviral transduction frequencies. This technique utilizes a conventional 96-well format and, when coupled with a fluorescence-based post-PCR detection system, makes it unnecessary to run agarose gels to visualize the PCR product. This system of PCR product detection, which uses the 5'-->3' exonuclease activity of Taq DNA polymerase to cleave a fluorescently labeled probe during each round of PCR amplification, is fast, convenient, and at least as sensitive as an ethidium bromide-based detection system when used in conjunction with our universal PCR assay.
We have recently demonstrated the geographic isolation of rice tungro bacilliform virus (RTBV) populations in the tungro-endemic provinces of Isabela and North Cotabato, Philippines. In this study, we examined the genetic structure of the virus populations at the tungro-outbreak sites of Lanao del Norte, a province adjacent to North Cotabato. We also analyzed the virus populations at the tungro-endemic sites of Subang, Indonesia, and Dien Khanh, Vietnam. Total DNA extracts from 274 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV. In the total population, 22 EcoRV-restricted genome profiles (genotypes) were identified. Although overlapping genotypes could be observed, the outbreak sites of Lanao del Norte had a genotype combination distinct from that of Subang or Dien Khanh but a genotype combination similar to that identified earlier from North Cotabato, the adjacent endemic province. Sequence analysis of the intergenic region and part of the ORF1 RTBV genome from randomly selected genotypes confirms the geographic clustering of RTBV genotypes and, combined with restriction analysis, the results suggest a fragmented spatial distribution of RTBV local populations in the three countries. Because RTBV depends on rice tungro spherical virus (RTSV) for transmission, the population dynamics of both tungro viruses were then examined at the endemic and outbreak sites within the Philippines. The RTBV genotypes and the coat protein RTSV genotypes were used as indicators for virus diversity. A shift in population structure of both viruses was observed at the outbreak sites with a reduced RTBV but increased RTSV gene diversity.
The genetic structure of rice tungro bacilliform virus (RTBV) populations within and between growing sites was analyzed in a collection of natural field isolates from different rice varieties grown in eight tungro-endemic sites of the Philippines. Total DNA extracts from 345 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV, a procedure shown in preliminary experiments capable of revealing high levels of polymorphism in RTBV field isolates. In the total population, 17 distinct EcoRV-based genome profiles (genotypes) were identified and used as indicators for virus diversity. Distinct sets of genotypes occurred in Isabela and North Cotabato provinces suggesting a geographic isolation of virus populations. However, among the sites in each province, there were few significant differences in the genotype compositions of virus populations. The number of genotypes detected at a site varied from two to nine with a few genotypes dominating. In general the isolates at a site persisted from season to season indicating a genetic stability for the local virus population. Over the sampling time, IRRI rice varieties, which have green leafhopper resistance genes, supported similar virus populations to those supported by other varieties, indicating that the variety of the host exerted no apparent selection pressures. Insect transmission experiments on selected RTBV field isolates showed that dramatic shifts in genotype and phenotype distributions can occur in response to host/environmental shifts.
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