Homologous Interference Research Articles

PNA-Functionalized, Silica Nanowires-Filled Glass Microtube for Ultrasensitive and Label-Free Detection of miRNA-21.

MicroRNAs (miRNAs) are endogenous and noncoding single-stranded RNA molecules with a length of approximately 18-25 nucleotides, which play an undeniable role in early cancer screening. Therefore, it is very important to develop an ultrasensitive and highly specific method for detecting miRNAs. Here, we present a bottom-up assembly approach for modifying glass microtubes with silica nanowires (SiNWs) and develop a label-free sensing platform for miRNA-21 detection. The three-dimensional (3D) networks formed by SiNWs make them abundant and highly accessible sites for binding with peptide nucleic acid (PNA). As a receptor, PNA has no phosphate groups and exhibits an overall electrically neutral state, resulting in a relatively small repulsion between PNA and RNA, which can improve the hybridization efficiency. The SiNWs-filled glass microtube (SiNWs@GMT) sensor enables ultrasensitive, label-free detection of miRNA-21 with a detection limit as low as 1 aM at a detection range of 1 aM-100 nM. Noteworthy, the sensor can still detect miRNA-21 in the range of 102-108 fM in complex solutions containing 1000-fold homologous interference of miRNAs. The high anti-interference performance of the sensor enables it to specifically recognize target miRNA-21 in the presence of other miRNAs and distinguish 1-, 3-mismatch nucleotide sequences. Significantly, the sensor platform is able to detect miRNA-21 in the lysate of breast cancer cell lines (e.g., MCF-7 cells and MDA-MB-231 cells), indicating that it has good potential in the screening of early breast cancers.

Cascade branch migration-triggered strand displacement amplification for specific and sensitive detection of microRNA.

MicroRNAs (miRNAs) have been involved in many biological processes and are regarded as promising biomarkers. The short sequence, low abundance and highly homologous interference sequences greatly hinder the accurate detection of miRNAs. Here, a cascade branch migration-triggered strand displacement amplification (CBM-TSDA) strategy was developed for the first time for specific and sensitive detection of miRNA-155 (miR-155). In the presence of target miR-155, the CBM was initiated and two Y-shaped probes were eventually produced. Next, the Y-shaped probes were transformed into three-way junction (3WJ) structures and triggered the SDA to produce a large number of G-quadruplex (G4) structures. Finally, the increased fluorescence signal of G4/Thioflavin T (ThT) was used to quantify miR-155. Meanwhile, the colorimetric responses of the G4-hemin DNAzyme could be used as supplementary detection to obtain a dual-mode signal readout. This detection strategy showed high detection sensitivity, and the limit of detection was 0.28 pM in the fluorescence detection mode and 0.34 pM in the colorimetric detection mode. Notably, it showed high detection specificity, being able to discriminate the single-base mutations of the target with a high discrimination factor. The strategy also possessed excellent capacity for miR-155 detection in cell lysates and real human blood samples. The developed strategy provides a promising detection platform for miRNA, which may be applied to early clinical diagnosis.

Spinal muscular atrophy type I associated with a novel SMN1 splicing variant that disrupts the expression of the functional transcript.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by pathogenic variants in the SMN1 gene. The majority of SMA patients harbor a homozygous deletion of SMN1 exon 7 (95%). Heterozygosity for a conventional variant and a deletion is rare (5%) and not easily detected, due to the highly homologous SMN2 gene interference. SMN2 mainly produces a truncated non-functional protein (SMN-d7) instead of the full-length functional (SMN-FL). We hereby report a novel SMN1 splicing variant in an infant with severe SMA. MLPA was used for SMN1/2 exon dosage determination. Sanger sequencing approaches and long-range PCR were employed to search for an SMN1 variant. Conventional and improved Real-time PCR assays were developed for the qualitative and quantitative SMN1/2 RNA analysis. The novel SMN1 splice-site variant c.835-8_835-5delinsG, was identified in compound heterozygosity with SMN1 exons 7/8 deletion. RNA studies revealed complete absence of SMN1 exon 7, thus confirming a disruptive effect of the variant on SMN1 splicing. No expression of the functional SMN1-FL transcript, remarkable expression of the SMN1-d7 and increased levels of the SMN2-FL/SMN2-d7 transcripts were observed. We verified the occurrence of a non-deletion SMN1 variant and supported its pathogenicity, thus expanding the SMN1 variants spectrum. We discuss the updated SMA genetic findings in the Cypriot population, highlighting an increased percentage of intragenic variants compared to other populations.

Characterization of Viral Interference in Aedes albopictus C6/36 Cells Persistently Infected with Dengue Virus 2.

Arboviruses are an important group of pathogens that cause diseases of medical and veterinary concern worldwide. The interactions of these viruses with their host cells are complex, and frequently, the coexistence of two different viruses in the same cell results in the inhibition of replication in one of the viruses, which is a phenomenon called viral interference. This phenomenon can be exploited to develop antiviral strategies. Insect cell lines persistently infected with arboviruses are useful models with which to study viral interference. In this work, a model of C6/36-HT cells (from Aedes albopictus mosquitoes) persistently infected with Dengue virus, serotype 2, was used. Viral interference was evaluated via plaque and flow cytometry assays. The presence of heterotypic interference against the other serotypes of the same virus and homologous interference against yellow fever virus was determined; however, this cell line did not display heterologous viral interference against Sindbis virus. The mechanisms responsible for viral interference have not been fully elucidated, but small RNAs could be involved. However, the silencing of Ago3, a key protein in the genome-derived P-element-induced wimpy testis pathway, did not alter the viral interference process, suggesting that viral interference occurs independent of this pathway.

Triple Amplification Strategy of CHA-PER-DNAzyme for Ultrasensitive Detection of circRNA Associated with Hepatocellular Carcinoma.

Biological and clinical studies have indicated that aberrant expression of circMTO1 served as a crucial biomarker for the diagnosis and prognosis of hepatocellular carcinoma (HCC) patients as well as a potential therapeutic target. However, the detection of circRNAs currently faces challenges such as homologous linear RNA interference and low-expression abundance of certain circRNAs. Therefore, we developed a triple amplification method based on catalytic hairpin assembly (CHA) activation by back-splice junction (BSJ), resulting in CHA products that triggered primer exchange reaction to generate DNAzyme. Subsequently, DNAzyme cleaved the fluorescent reporter chain, enabling ultrasensitive detection of hepatocellular carcinoma-associated circMTO1 through the output fluorescence signal. The catalytic hairpin opening sequence within CHA specifically targeted the BSJ sequence in circRNA, thereby avoiding false positive signals observed in circRNA assays due to the recognition of homologous linear RNA molecules. Moreover, this triple amplification method facilitated sensitive detection of circRNA and addressed the issue of low-abundance expression levels associated with circMTO1 in HCC samples. Notably, our newly designed assay for detecting circRNA exhibited a linear range from 1 fM to 100 nM with a detection limit of 0.265 fM. Furthermore, it demonstrated excellent and consistent performance even within complex systems. Our proposed method enabled the specific and sensitive detection of circMTO1 in various cancer cells and blood samples from HCC patients, providing an innovative approach for investigating the role of circRNA in tumorigenesis and development while promoting its clinical application.

Establishment of RNA Interference Genetic Transformation System and Functional Analysis of FlbA Gene in Leptographium qinlingensis.

Leptographium qinlingensis is a pathogenic fungus of Pinus armandii that is epidemic in the Qinling Mountains. However, an effective gene interference strategy is needed to characterize the pathogenic genes in this fungus on a functional level. Using the RNA silencing vector pSilent-1 as a template, we established an RNA interference genetic transformation system mediated by Agrobacterium tumefaciens GV3101, which is suitable for the gene study for Leptographium qinlingensis by homologous recombination and strain interference system screening. The LqFlbA gene was silenced using the RNA interference approach described above, and the resulting transformants displayed various levels of silencing with a gene silencing effectiveness ranging from 41.8% to 91.4%. The LqFlbA-RNAi mutant displayed altered colony morphology, sluggish mycelium growth, and diminished pathogenicity toward the host P. armandii in comparison to the wild type. The results indicate that this method provides a useful reverse genetic system for studying the gene function of L. qinlingensis, and that LqFlbA plays a crucial role in the growth, development, and pathogenicity of L. qinlingensis.

Bifunctional probe-assisted hybridization chain reaction for circular RNA detection and live-cell imaging

Circular RNA (circRNA) is considered as a promising biomarker for various diseases owing to its unique functions and stable structure in biological circumstances. Nonetheless, homologous linear RNA always causes detection errors, posing a key challenge in effective circRNA assays for clinical applications. Herein, we present a novel method to directly analyze circRNA, eliminating homologous linear RNA interference. A dual-function circRNA-identifying hairpin (ih) probe is designed, which identifies target circRNA and initiates the subsequent hybridization chain reaction amplification. This method provides highly sensitive live-cell circRNA imaging capability and can directly monitor circRNA levels in biological samples, showing great promise for the analysis of low-abundant circRNA in clinical applications.

Destruction of the vascular viral receptor in infectious salmon anaemia provides in vivo evidence of homologous attachment interference.

Viral interference is a process where infection with one virus prevents a subsequent infection with the same or a different virus. This is believed to limit superinfection, promote viral genome stability, and protect the host from overwhelming infection. Mechanisms of viral interference have been extensively studied in plants, but remain poorly understood in vertebrates. We demonstrate that infection with infectious salmon anaemia virus (ISAV) strongly reduces homologous viral attachment to the Atlantic salmon, Salmo salar L. vascular surface. A generalised loss of ISAV binding was observed after infection with both high-virulent and low-virulent ISAV isolates, but with different kinetics. The loss of ISAV binding was accompanied by an increased susceptibility to sialidase, suggesting a loss of the vascular 4-O-sialyl-acetylation that mediates ISAV attachment and simultaneously protects the sialic acid from cleavage. Moreover, the ISAV binding capacity of cultured cells dramatically declined 3 days after ISAV infection, accompanied by reduced cellular permissiveness to infection with a second antigenically distinct isolate. In contrast, neither infection with infectious haematopoietic necrosis virus nor stimulation with the viral mimetic poly I:C restricted subsequent cellular ISAV attachment, revealing an ISAV-specific mechanism rather than a general cellular antiviral response. Our study demonstrates homologous ISAV attachment interference by de-acetylation of sialic acids on the vascular surface. This is the first time the kinetics of viral receptor destruction have been mapped throughout the full course of an infection, and the first report of homologous attachment interference by the loss of a vascular viral receptor. Little is known about the biological functions of vascular O-sialyl-acetylation. Our findings raise the question of whether this vascular surface modulation could be linked to the breakdown of central vascular functions that characterises infectious salmon anaemia.

In silico evaluation of molecular virus-virus interactions taking place between Cotton leaf curl Kokhran virus- Burewala strain and Tomato leaf curl New Delhi virus.

BackgroundCotton leaf curl disease (CLCuD) is a disease of cotton caused by begomoviruses, leading to a drastic loss in the annual yield of the crop. Pakistan has suffered two epidemics of this disease leading to the loss of billions in annual exports. The speculation that a third epidemic of CLCuD may result as consequence of the frequent occurrence of Tomato leaf curl New Delhi virus (ToLCNDV) and Cotton leaf curl Kokhran Virus-Burewala Strain (CLCuKoV-Bu) in CLCuD infected samples, demand that the interactions taking between the two viruses be properly evaluated. This study is designed to assess virus-virus interactions at the molecular level and determine the type of co-infection taking place.MethodsBased on the amino acid sequences of the gene products of both CLCuKoV-Bu and ToLCNDV, protein structures were generated using different software, i.e., MODELLER, I-TASSER, QUARKS, LOMETS and RAPTORX. A consensus model for each protein was selected after model quality assessment using ERRAT, QMEANDisCo, PROCHECK Z-Score and Ramachandran plot analysis. The active and passive residues in the protein structures were identified using the CPORT server. Protein–Protein Docking was done using the HADDOCK webserver, and 169 Protein–Protein Interaction (PPIs) were performed between the proteins of the two viruses. The docked complexes were submitted to the PRODIGY server to identify the interacting residues between the complexes. The strongest interactions were determined based on the HADDOCK Score, Desolvation energy, Van der Waals Energy, Restraint Violation Energy, Electrostatic Energy, Buried Surface Area and Restraint Violation Energy, Binding Affinity and Dissociation constant (Kd). A total of 50 ns Molecular Dynamic simulations were performed on complexes that exhibited the strongest affinity in order to validate the stability of the complexes, and to remove any steric hindrances that may exist within the structures.ResultsOur results indicate significant interactions taking place between the proteins of the two viruses. Out of all the interactions, the strongest were observed between the Replication Initiation protein (Rep) of CLCuKoV-Bu with the Movement protein (MP), Nuclear Shuttle Protein (NSP) of ToLCNDV (DNA-B), while the weakest were seen between the Replication Enhancer protein (REn) of CLCuKoV-Bu with the REn protein of ToLCNDV. The residues identified to be taking a part in interaction belonged to domains having a pivotal role in the viral life cycle and pathogenicity. It maybe deduced that the two viruses exhibit antagonistic behavior towards each other, and the type of infection may be categorised as a type of Super Infection Exclusion (SIE) or homologous interference. However, further experimentation, in the form of transient expression analysis, is needed to confirm the nature of these interactions and increase our understanding of the direct interactions taking place between two viruses.

Quantitative and rapid interference of bovine viral diarrhea virus BVDV/END- and BVDV/END+ strains.

Homologous interference in vitro is defined as the ability of primary viral infection to prevent secondary homologous superinfection. Non-cytopathic bovine viral diarrhea virus (ncp BVDV) has been classified according to the exaltation of Newcastle disease phenomenon (END) as END positive (E+) and END negative (E-) strains. Simultaneous inoculation of MDBK-SY cell monolayers with BVDV/E- virus and a three log higher amount of BVDV RK13/E+ virus, leads to acquisition of the BVDV/E- feature of blocking Newcastle disease virus (NDV) infection in cells. BVDV/E- strains, particularly at a high titre and MOI ≥1.25, can exert and impose their effects in BVDV/E+ infected cells; however, if BVDV/E- MOI is reduced to MOI below 0.625, the BVDV/E+ effect can be restored leading to cytopathic effects (CPE) induction by NDV reciprocal to the titre of the BVDV RK13/E+ strain. Moreover, blocking and prevention of induced CPE by NDV or vesicular stomatitis virus (VSV) occurs even when BVDV/E- superinfects primary BVDV/E+ infected cells, indicating a defective homologous interference between BVDV/E+ and BVDV/E- strains. Taken together, BVDV/E- strains have a strong competitive potency and mediate a fast acting (i.e. within 60 min) influence against BVDV/E+ activity. This may be relevant in vivo where BVDV/E- and BVDV/E+ combinations are frequently isolated from infected individuals.

Random mutagenesis and precise gene editing technologies: applications in algal crop improvement and functional genomics

ABSTRACTThe establishment of a system for gene modification is crucial for the generation of new improved algal strains and elucidation of functional genome organization to enhance our understanding of algal biology. Several gene transfer methods have been developed for stable introduction of transgenes into algae allowing expression of desired foreign proteins. Site-specific gene integration and gene knockdown were achieved through homologous recombination and RNA interference approaches. The nuclease-associated gene editing technologies such as CRISPR-associated RNA-guided endonuclease Cas9 (CRISPR-Cas9) could efficiently generate stable targeted gene editing in algae. Although gene modification technologies have been established for algae, there are still practical difficulties that need to be addressed prior to commercialization such as transgene stability, potential risks and public acceptance. Genetic mitigation and containment strategies should be considered for commercial-scale production of transgenic algae.Abbreviations: ALE, adaptive laboratory evolution; AmiRNA, artificial microRNA; BER, base excision repair; CaMV35S, Cauliflower Mosaic Virus 35S; Cas9, CRISPR-associated protein 9; Cas9n, Cas9 mutant nickase; CRISPR, clustered, regularly interspaced, short palindromic repeats; crRNA, CRISPR RNA; dCAS9, nuclease-deficient Cas9, dead Cas9 or inactive Cas9; DSB, double-stranded break; GM, genetically modified; HDR, homology-directed repair; HR, homologous recombination; miRNA, microRNA; mRNA, messenger RNA; NHEJ, non-homologous end joining; NiR, nitrite reductase; NR, nitrate reductase; PAM, Protospacer-Adjacent Motif; RNAi, RNA interference; sgRNA, single guide RNA; siRNA, small interfering RNA; sRNA, small RNA; SV40, Simian vacuolating virus 40 or Simian virus 40; TALEN, transcription-activator like effector nuclease; tracrRNA, transactivating CRISPR RNA; UTR, Untranslated region; ZFN, zinc-finger nuclease.

A unified approach towards Trypanosoma brucei functional genomics using Gibson assembly

Trypanosoma brucei is the causative agent of human African trypanosomiasis and nagana in cattle. Recent advances in high throughput phenotypic and interaction screens have identified a wealth of novel candidate proteins for diverse functions such as drug resistance, life cycle progression, and cytoskeletal biogenesis. Characterization of these proteins will allow a more mechanistic understanding of the biology of this important pathogen and could identify novel drug targets. However, methods for rapidly validating and prioritizing these potential targets are still being developed. While gene tagging via homologous recombination and RNA interference are available in T. brucei, a general strategy for creating the most effective constructs for these approaches is lacking. Here, we adapt Gibson assembly, a one-step isothermal process that rapidly assembles multiple DNA segments in a single reaction, to create endogenous tagging, overexpression, and long hairpin RNAi constructs that are compatible with well-established T. brucei vectors. The generality of the Gibson approach has several advantages over current methodologies and substantially increases the speed and ease with which these constructs can be assembled.

Evidence that Receptor Destruction by the Sendai Virus Hemagglutinin-Neuraminidase Protein Is Responsible for Homologous Interference.

Receptor destruction has been considered one of the mechanisms of homologous Sendai virus (SeV) interference. However, direct evidence of receptor destruction upon virus infection and its relevance to interference is missing. To investigate a precise mechanism of homologous interference, we established SeV persistently infected cells. The persistently infected cells inhibited superinfection by homologous SeV but supported replication of human parainfluenza virus 2 (hPIV2) and influenza A virus (IAV). We confirmed that SeV particles could not attach to or penetrate the infected cells and that the hemagglutinin-neuraminidase (HN) protein of SeV was involved in the interference. Lectin blot assays showed that the α2,3-linked sialic acids were specifically reduced in the SeV-infected cells, but the level of α2,6-linked sialic acids had not changed. As infection with IAV removed both α2,3- and α2,6-linked sialic acids, especially α2,3-linked sialic acids, IAV-infected cells inhibited superinfection of SeV. These results provide concrete evidence that destruction of the specific SeV receptor, α2,3-linked sialic acids, is relevant to homologous interference by SeV. Viral interference is a classically observed phenomenon, but the precise mechanism is not clear. Using SeV interference, we provide concrete evidence that reduction of the α2,3-linked sialic acid receptor by the HN of SeV is closely related with viral interference. Since SeV infection resulted in decrease of only α2,3-linked sialic acids, IAV, which also utilized α2,6-linked sialic acids to initiate infection, superinfected the SeV-infected cells. In contrast, SeV could not superinfect the IAV-infected cells because both α2,3- and α2,6-linked sialic acids were removed. These results indicate that receptor destruction critically contributes to viral interference.

Eilat virus induces both homologous and heterologous interference

Most alphaviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates including birds, rodents, equids, and humans. Occasionally, alphaviruses can spill over into the human population and cause disease characterized by debilitating arthralgia or fatal encephalitis. Recently, a unique alphavirus, Eilat virus (EILV), was described that readily infects mosquito but not vertebrate cell lines. Here, we investigated the ability of EILV to induce superinfection exclusion. Prior infection of C7/10 (Aedes albopictus) cells with EILV induced homologous and heterologous interference, reducing the virus titers of heterologous superinfecting viruses (SINV, VEEV, EEEV, WEEV, and CHIKV) by ~10–10,000 fold and delaying replication kinetics by 12–48h. Similar to in vitro infection, prior in vivo EILV infection of Aedes aegypti mosquitoes delayed dissemination of chikungunya virus for 3 days. This is the first evidence of heterologous interference induced by a mosquito-specific alphavirus in vitro and in vivo.

Superinfection Exclusion in Alphabaculovirus Infections Is Concomitant with Actin Reorganization

Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed, but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection). Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses--AcMNPV and SfMNPV--but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses. Infection of a cell by more than one virus particle implies sharing of cell resources. We show that multiple infection, by closely related or distantly related baculoviruses, is possible only during a brief window of time that allows additional virus particles to enter an infected cell over a period of ca. 16 h but then blocks multiple infections as newly generated virus particles begin to leave the infected cell. This temporal window has two important consequences. First, it allows multiple genotypes to almost simultaneously infect cells within the host, thus generating genetically diverse virus particles for transmission. Second, it provides a mechanism by which different viruses replicating in the same cell nucleus can exchange genetic material, so that the progeny viruses may be a mosaic of genes from each of the parental viruses. This opens a completely new avenue of research into the evolution of these insect pathogens.

Hypervaccination as Prompt Non-immune Protection

Hypervaccination against infectious diseases is a radically new efficient strategy of preventive vaccination. Its objective is specific abrogation of innate susceptibility of the organism to the disease achieved by blockage of the pathogen specific binding sites (receptors) on the surface of target cells by the anti-receptors of the vaccine. The new strategy is based on competition between the anti-receptors of the vaccine and those of the pathogen for the receptors on target cells. The mechanism of hypervaccination is a rapid maximal saturation of the target cells receptors with a massive dose of anti-receptors contained in a live or inactivated vaccine. Protection of the naturally susceptible organism against infection initially occurs by the mechanism of homologous interference at the stage of the pathogen's binding to the target cell rather than by reactions of adaptive immunity. At a later stage, early protection is followed by a long-term intense immune response with a marked seroconversion. Thus, a high dose vaccination confers protection in two stages, interference at the receptor binding level and immune response. Administering a safe vaccine with a high content of anti-receptors protects naturally susceptible animals against disease and death at challenge 12-72hrs after vaccination with no chronic infection establishing. An apparent resistance of the organism against a severe challenge soon after vaccination is likely to be due to a full or virtually full saturation of the target cells receptors with the vaccine anti-receptors. The efficiency of prompt protection in field conditions has first been shown in the eradication of classical swine fever (CSF) in two countries, Russia and Belarus.

Crossover rate between homologous chromosomes and interference are regulated by the addition of specific unpaired chromosomes in Brassica

Recombination is a major mechanism generating genetic diversity, but the control of the crossover rate remains a key question. In Brassica napus (AACC, 2n = 38), we can increase the homologous recombination between A genomes in AAC hybrids. Hypotheses for this effect include the number of C univalent chromosomes, the ratio between univalents and bivalents and, finally, which of the chromosomes are univalents. To test these hypotheses, we produced AA hybrids with zero, one, three, six or nine additional C chromosomes and four different hybrids carrying 2n = 32 and 2n = 35 chromosomes. The genetic map lengths for each hybrid were established to compare their recombination rates. The rates were 1.4 and 2.7 times higher in the hybrids having C6 or C9 alone than in the control (0C). This enhancement reached 3.1 and 4.1 times in hybrids carrying six and nine C chromosomes, and it was also higher for each pair of hybrids carrying 2n = 32 or 2n = 35 chromosomes, with a dependence on which chromosomes remained as univalents. We have shown, for the first time, that the presence of one chromosome, C9 , affects significantly the recombination rate and reduces crossover interference. This result will have fundamental implications on the regulation of crossover frequency.

A coat-independent superinfection exclusion rapidly imposed in Nicotiana benthamiana cells by tobacco mosaic virus is not prevented by depletion of the movement protein

New evidence is emerging which indicates that population variants in plant virus infections are not uniformly distributed along the plant, but structured in a mosaic-like pattern due to limitation to the superinfection imposed by resident viral clones. The mechanisms that prevent the infection of a challenge virus into a previously infected cell, a phenomenon known as superinfection exclusion (SE) or Homologous Interference, are only partially understood. By taking advantage of a deconstructed tobacco mosaic virus (TMV) system, where the capsid protein (CP) gene is replaced by fluorescent proteins, an exclusion mechanism independent of CP was unveiled. Time-course superinfection experiments provided insights into SE dynamics. Initial infection levels affecting less than 10% of cells led to full immunization in only 48h, and measurable immunization levels were detected as early as 6h post-primary infection. Depletion of a functional movement protein (MP) was also seen to slow down, but not to prevent, the SE mechanism. These observations suggest a CP-independent mechanism based on competition for a host-limiting factor, which operates at very low virus concentration. The possible involvement of host factors in SE has interesting implications as it would enable the host to influence the process.

Competitive virus assay method for titration of noncytopathogenic bovine viral diarrhea viruses (END+ and END− viruses)

A new, reliable and secure virus assay method, named the competitive virus assay (CVA) method, has been established for the titration of bovine viral diarrhea viruses (BVDVs) that either show the exaltation of Newcastle disease virus (END) phenomenon or heterologous interference phenomenon (but not the END phenomenon). This method is based on the principle of (1) homologous interference between BVDVs, by using BVDV RK13/E(-) or BVDV RK13/E(+) strains as competitor virus, and (2) END phenomenon and heterologous interference, by using attenuated Newcastle disease virus (NDV) TCND strain as challenge virus. In titration of BVDV END(+) and BVDV END(-) viruses, no significant difference in estimated virus titer was observed between CVA and conventional methods. CVA method demonstrated comparable levels of sensitivity and accuracy as conventional END and interference methods, which require the use of a velogenic Miyadera strain of NDV and vesicular stomatitis virus (VSV), both of which are agents of high-risk diseases. As such, the CVA method is a safer alternative, with increased bio-safety and bio-containment, through avoidance of virulent strains that are commonly employed with conventional methods.

Using mutants, knockdowns, and transgenesis to investigate gene function in Drosophila

AbstractThe sophisticated genetic techniques available in Drosophila are largely responsible for its success as a model organism. One of the most important of these is the ability to disrupt gene function in vivo and observe the resulting phenotypes. This review considers the ever‐increasing repertoire of approaches for perturbing the functions of specific genes in flies, ranging from classical and transposon‐mediated mutageneses to newer techniques, such as homologous recombination and RNA interference. Since most genes are used over and over again in different contexts during development, many important advances have depended on being able to interfere with gene function at specific times or places in the developing animal, and a variety of approaches are now available to do this. Most of these techniques rely on being able to create genetically modified strains of Drosophila and the different methods for generating lines carrying single copy transgenic constructs will be described, along with the advantages and disadvantages of each approach. WIREs Dev Biol 2013, 2:587–613. doi: 10.1002/wdev.101This article is categorized under: Technologies > Perturbing Genes and Generating Modified Animals Technologies > Generating Chimeras and Lineage Analysis Technologies > Analysis of Cell, Tissue, and Animal Phenotypes