Standardization of denaturing gradient gel electrophoresis for mutant screening of influenza A (H3N2) virus samples

Motta, Fernando Couto; Rosado, Alexandre Soares; Couceiro, J. N. S. S.

doi:10.1016/s0166-0934(01)00426-8

Cited by 12 publications

(7 citation statements)

References 31 publications

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“…DGGE is an extremely sensitive molecular technique capable of detecting SNPs within DNA fragments due to changes in their melting characteristics [61,62]. Since DGGE has been utilized successfully in the detection of genetic variation in a number of viral systems [23,63,64], we developed DGGE assays for the dbp1, DNA polymerase and me53 genes to provide an additional method for evaluating genetic variation and provide evidence in support of the NGS findings. The five DGGE assays were clearly capable of detecting a large number of genetic variants within the HearNPV populations, with many bands present for each primer set and each band representing a slightly different DNA sequence.…”

Section: Discussionmentioning

confidence: 99%

“…However, RFLP analysis has several innate limitations; it can only detect polymorphisms present within the enzyme recognition sites, it is a hit and miss approach with regard to the enzymes chosen to digest the DNA, and it cannot show what the polymorphisms are or where the polymorphisms occur within the genomes. These limitations restrict the usefulness of RFLP for the study of variation within virus genomes [21][22][23].…”

Section: Introductionmentioning

confidence: 99%

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High levels of genetic variation within core Helicoverpa armigera nucleopolyhedrovirus genes

Baillie

Bouwer

2011

Virus Genes

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It is well documented that baculovirus populations contain many genotypic variants, but little is known about the degree of genetic variation in specific baculovirus genes. Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was used as a model system for studying genetic variation in nucleopolyhedrovirus genes. Next generation sequencing (NGS) was used to identify single-nucleotide polymorphisms (SNPs) within a core baculovirus gene (DNA polymerase) and two core lepidopteran-specific baculovirus genes (dbp1 and me53) in HearNPV populations. Analysis of the NGS data identified 60 SNPs within the 3063 bp DNA polymerase gene, 13 SNPs within the 972 bp dbp1 gene, and 25 SNPs within the 1080 bp me53 gene. Depending on the gene, between 31 and 35% of the SNPs were non-synonymous and may, thus, affect the biological functioning of the encoded proteins. The number and homogenous distribution of SNPs found suggest that nucleotide substitution is a major contributor to HearNPV genetic diversity. Denaturing gradient gel electrophoresis (DGGE) assays were used to provide an additional method for evaluating HearNPV genetic variation. Since each of the gene-specific DGGE assays produced unique banding profiles containing numerous bands of differing relative intensities, the DGGE experiments confirmed that there was a high degree of genetic variation in core HearNPV genes and provided information on the relative frequency distribution of genetic variants in the population. The amount of gene-specific genetic variation detected in this study significantly exceeds that reported in other HearNPV studies, suggesting that NGS and DGGE may be useful techniques for studying genetic variation in other baculoviruses.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

High levels of genetic variation within core Helicoverpa armigera nucleopolyhedrovirus genes

Baillie

Bouwer

2011

Virus Genes

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“…Baillie & Bouwer (2011) showed that denaturing gradient gel electrophoresis (DGGE) was capable of detecting genetic variants within geographically distinct Helicoverpa armigera nucleopolyhedrovirus (HearNPV) populations in a highly sensitive and rapid manner. DGGE has also been used to study the population dynamics of viral communities as well as to study how the populations respond to changes in environmental and experimental conditions (Harris & Teo, 2001;Motta et al, 2002;Short & Suttle, 1999).…”

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confidence: 99%

The effect of inoculum dose on the genetic diversity detected within Helicoverpa armigera nucleopolyhedrovirus populations

Baillie

Bouwer

2013

Journal of General Virology

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Environmental and infection variables may affect the genetic diversity of baculovirus populations. In this study, Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was used as a model system for studying the effects of a key infection variable, inoculum dose, on the genetic diversity within nucleopolyhedrovirus populations. Diversity and equitability indices were calculated from DNA polymerase-specific denaturing gradient gel electrophoresis profiles obtained from individual H. armigera neonate larvae inoculated with either an LD 5 or LD 95 of HearNPV. Although the genetic diversity detected in larvae treated with an LD 95 was not statistically different from the diversity detected in the HearNPV inoculum samples, there was a statistically significant difference in the genetic diversity detected in the LD 5 -inoculated larvae compared with the genetic diversity detected in the HearNPV samples used for the inoculations. The study suggests that inoculum dose needs to be considered carefully in experiments that evaluate HearNPV genetic diversity or in studies where differences in genetic diversity may have phenotypic consequences.Baculoviruses are dsDNA viruses that are characterized by proteinaceous occlusion bodies (OBs) that enclose the virions (Funk et al., 1997;Theilmann & Blissard, 2008). The family Baculoviridae is divided into four genera (Alphabaculovirus, Betabaculovirus, Deltabaculovirus and Gammabaculovirus) based on phylogenetics, genome composition, host range, and morphological and pathological traits (Jehle et al., 2006). The alphabaculoviruses include the lepidopteran-specific nucleopolyhedroviruses (NPVs), which have virions that may contain a single nucleocapsid (SNPV) or multiple nucleocapsids (MNPV) (Theilmann & Blissard, 2008).Baculoviruses have been shown to have a high degree of genotypic variation between and within baculovirus isolates (Cory et al., 2005;Crook et al., 1985;Graham et al., 2004; Hitchman et al., 2007; Parnell et al., 2002;van Oers & Vlak, 2007). It has even been shown that baculovirus genetic variation can be detected within an individual host insect (Baillie & Bouwer, 2012b;Cory et al., 2005). Furthermore, Cory et al. (2005) showed that a number of the genetic variants isolated from a single host insect differed in phenotypic traits, including those for pathogenicity and kill time.The maintenance of genetic variation may be affected by host immune response specificity, tradeoffs between virus genotype fitness components, interactions of genotypes within the host, and differential selection of genotypes (Hitchman et al., 2007;Hodgson et al., 2001Hodgson et al., , 2002. Genetic heterogeneity must be important for the survival of baculoviruses as the less dominant (minority) viral variants are not lost from the virus population over time (Simó n et al., 2008). It has been shown that the minority genotypes affect the timing of mortality and the overall pathogenicity of the virus population (Simó n et al., 2005(Simó n et al., , 2008. The mixture of genotypes may consist of c...

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“…In terms of practicality and cost, nucleic acid sequencing is still not indicated as the first technique to be used for searching for differences among nucleic acid sequences when there is a considerable number of samples to be analyzed (Motta et al 2002). Several techniques have been developed to search for DNA mutations prior to sequencing, and this approach may reduce the number of samples to be sequenced (Gouvea et al 1993, Motta et al 2006).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, large DNA molecules may be cleaved into fragments <1Kb in size that often present the best DGGE results, or may be analyzed by restriction fragment length polymorphism (RFLP) combined with DGGE (Sheffield et al 1989). No such tools are available for dsRNA, unless they are converted into cDNA copies by means of RT-PCR and analyzed as DNA molecules, which is the approach most frequently taken (Gouvea et al 1993, Motta et al 2002. Here, we demonstrated that DGGE and CDGE techniques may be used to screen for mutations or to study polymorphisms directly in segmented dsRNA virus genomes.…”

Section: Discussionmentioning

confidence: 99%

DETECTION OF MUTATIONS IN AVIAN REOVIRUS dsRNA GENOME BY DENATURING GRADIENT (DGGE) AND CONSTANT DENATURING (CDGE) GEL ELECTROPHORESIS

Naveca¹,

Domingues²,

Gouvêa³

2007

VR&R

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Standardization of denaturing gradient gel electrophoresis for mutant screening of influenza A (H3N2) virus samples

Cited by 12 publications

References 31 publications

High levels of genetic variation within core Helicoverpa armigera nucleopolyhedrovirus genes

High levels of genetic variation within core Helicoverpa armigera nucleopolyhedrovirus genes

The effect of inoculum dose on the genetic diversity detected within Helicoverpa armigera nucleopolyhedrovirus populations

DETECTION OF MUTATIONS IN AVIAN REOVIRUS dsRNA GENOME BY DENATURING GRADIENT (DGGE) AND CONSTANT DENATURING (CDGE) GEL ELECTROPHORESIS

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