Reverse Genetics Research Articles

N-glycosylation of the envelope glycoprotein I is essential for the proliferation and virulence of the duck plague virus

Duck plague virus (DPV) causes the highly pathogenic duck plague, and the envelope glycoprotein I (gI), as one of the key virulence genes, has not yet had its critical virulence sites identified through screening. This study used reverse genetics technology to target the gI, specifically within the DPV genome. Four DPV mutants with gI N-glycosylation site mutations were designed and constructed, and these mutant strains were successfully rescued. Our results confirmed that three asparagine residues of gI (N69, N78, and N265) are N-glycosylation sites, and western blot analysis substantiated that glycosylation at each predicted N-glycosylation site was compromised. The deglycosylation of gI leads to the protein misfolding and subsequent retention in the endoplasmic reticulum (ER). The subsequent deglycosylated gI is carried into the Golgi apparatus (GM130) in the interaction of gE. Compared to the parental virus, the mutated virus shows a 66.3% reduction in intercellular transmission capability. In ducks, the deglycosylation of gI significantly reduces DPV replication in vivo, thereby weakening the virulence of DPV. This study represents the first successful creation of a weak DPV virus strain by specific mutation at the N-glycosylation site. The findings provide a foundational understanding of DPV pathogenesis and form the basis for developing live attenuated vaccines against the disease.

Expanding the BonnMu sequence-indexed repository of transposon induced maize (Zea mays L.) mutations in dent and flint germplasm.

The BonnMu resource is a transposon tagged mutant collection designed for functional genomics studies in maize. To expand this resource, we crossed an active Mutator (Mu) stock with dent (B73, Co125) and flint (DK105, EP1, and F7) germplasm, resulting in the generation of 8064 mutagenized BonnMu F2-families. Sequencing of these Mu-tagged families revealed 425 924 presumptive heritable Mu insertions affecting 36 612 (83%) of the 44 303 high-confidence gene models of maize (B73v5). On average, we observed 12 Mu insertions per gene (425 924 total insertions/36 612 affected genes) and 53 insertions per BonnMu F2-family (425 924 total insertions/8064 families). Mu insertions and photos of seedling phenotypes from segregating BonnMu F2-families can be accessed through the Maize Genetics and Genomics Database (MaizeGDB). Downstream examination via the automated Mutant-seq Workflow Utility (MuWU) identified 94% of the presumptive germinal insertion sites in genic regions and only a small fraction of 6% inserting in non-coding intergenic sequences of the genome. Consistently, Mu insertions aligned with gene-dense chromosomal arms. In total, 42% of all BonnMu insertions were located in the 5' untranslated region of genes, corresponding to accessible chromatin. Furthermore, for 38% of the insertions (163 843 of 425 924 total insertions) Mu1, Mu8 and MuDR were confirmed to be the causal Mu elements. Our publicly accessible European BonnMu resource has archived insertions covering two major germplasm groups, thus facilitating both forward and reverse genetics studies.

Two amino acid residues in the N-terminal region of the polymerase acidic protein determine the virulence of Eurasian avian-like H1N1 swine influenza viruses in mice.

Reassortant Eurasian avian-like H1N1 (rEA H1N1) viruses carrying the internal genes of H1N1/2009 virus have been circulating in pigs for more than 10 years and have caused sporadic human infections. The enhanced virulence phenotype of the rEA H1N1 viruses highlights potential risks to public health. However, the molecular mechanism underlying the viral pathogenicity of the currently circulating rEA H1N1 viruses remains unclear. In this study, we found that two naturally isolated rEA H1N1 swine influenza viruses, A/swine/Liaoning/FX38/2017 (FX38) and A/swine/Liaoning/SY72/2018 (SY72), possessed similar genetic characteristics but exhibited significantly different pathogenicity in a mouse model. Using reverse genetics, we demonstrated that amino acid mutations at positions 100 and 122 in the polymerase acidic (PA) protein had individual and synergistic effects on the polymerase activity and viral replication capacity in vitro, as well as the viral pathogenicity in mice. Furthermore, we revealed that amino acid residue 100 in PA influenced the transcription of viral RNA (vRNA) by altering the endonuclease activity, and amino acid residue 122 affected the synthesis of complementary RNA and messenger RNA by altering the RNA-binding ability and endonuclease activity of the PA protein. Taken together, we identified that two naturally occurring amino acid mutations in PA derived from H1N1/2009 virus are crucial determinants of the virulence of rEA H1N1 viruses and revealed the differential mechanism by which these two mutations affect the transcription and replication of vRNA. These findings will extend our understanding of the roles of PA in the virulence of influenza A viruses.IMPORTANCEMultiple genetic determinants are involved in the virulence of influenza A viruses. In this study, we identified two naturally occurring amino acid mutations, located at residues 100 and 122 in the polymerase acidic (PA) protein, which are associated with viral polymerase activity, replication competence, and pathogenicity in mice. In particular, we clarified the specific mechanism by which the two residues play an important role in viral transcription and replication. These findings will help to improve understanding the functions of amino acid residues in the N-terminal region of the PA protein involved in the pathogenicity of influenza A viruses.

Zika viruses encode 5′ upstream open reading frames affecting infection of human brain cells

Zika virus (ZIKV), an emerging mosquito-borne flavivirus, is associated with congenital neurological complications. Here, we investigate potential pathological correlates of virus gene expression in representative ZIKV strains through RNA sequencing and ribosome profiling. In addition to the single long polyprotein found in all flaviviruses, we identify the translation of unrecognised upstream open reading frames (uORFs) in the genomic 5′ region. In Asian/American strains, ribosomes translate uORF1 and uORF2, whereas in African strains, the two uORFs are fused into one (African uORF). We use reverse genetics to examine the impact on ZIKV fitness of different uORFs mutant viruses. We find that expression of the African uORF and the Asian/American uORF1 modulates virus growth and tropism in human cortical neurons and cerebral organoids, suggesting a potential role in neurotropism. Although the uORFs are expressed in mosquito cells, we do not see a measurable effect on transmission by the mosquito vector in vivo. The discovery of ZIKV uORFs sheds new light on the infection of the human brain cells by this virus and raises the question of their existence in other neurotropic flaviviruses.

Enhanced Oncolytic Potential of Engineered Newcastle Disease Virus Lasota Strain through Modification of Its F Protein Cleavage Site.

Newcastle disease virus (NDV) is an oncolytic virus whose F protein cleavage activity is associated with viral infectivity. To explore the potential of modifying F protein cleavage activity to enhance antitumor effects, we constructed a recombinant NDV LaSota strain by replacing its F protein cleavage site with that from the mesogenic Beaudette C (BC) strain using reverse genetics techniques. The resulting virus, rLaSota-BC-RFP, demonstrated significantly enhanced infectivity and tumor cell suppression on the murine melanoma B16F10 cell, characterized by higher cytotoxicity and increased apoptosis compared to its parental strain, rLaSota-RFP. In vivo, rLaSota-BC-RFP treatment of B16F10 tumors in C57BL/6 mice resulted in significant tumor growth inhibition, improved survival rate, and induction of tumor-specific apoptosis and necrosis. Additionally, the rLaSota-BC-RFP treatment enhanced immunostimulatory effects within the tumor microenvironment (TME), characterized by increased infiltration of CD4+ and CD8+ T cells and elevated levels of antitumor immune modulator cytokines, including mouse IL-12, IFN-γ, IL-15, and TNF-α, in the rLaSota-BC-RFP-treated tumor tissues. Collectively, these findings demonstrate that the mesogenic F protein cleavage site enhances the oncolytic potential of the NDV LaSota strain, suggesting that rLaSota-BC-RFP is a promising oncolytic viral vector for gene delivery in cancer immunotherapy.

Recombinant Influenza A Viruses Expressing Reporter Genes from the Viral NS Segment.

Studying influenza A viruses (IAVs) requires secondary experimental procedures to detect the presence of the virus in infected cells or animals. The ability to generate recombinant (r)IAV using reverse genetics techniques has allowed investigators to generate viruses expressing foreign genes, including fluorescent and luciferase proteins. These rIAVs expressing reporter genes have allowed for easily tracking viral infections in cultured cells and animal models of infection without the need for secondary approaches, representing an excellent option to study different aspects in the biology of IAV where expression of reporter genes can be used as a readout of viral replication and spread. Likewise, these reporter-expressing rIAVs provide an excellent opportunity for the rapid identification and characterization of prophylactic and/or therapeutic approaches. To date, rIAV expressing reporter genes from different viral segments have been described in the literature. Among those, rIAV expressing reporter genes from the viral NS segment have been shown to represent an excellent option to track IAV infection in vitro and in vivo, eliminating the need for secondary approaches to identify the presence of the virus. Here, we summarize the status on rIAV expressing traceable reporter genes from the viral NS segment and their applications for in vitro and in vivo influenza research.

Rhesus rotavirus NSP1 mediates extra-intestinal infection and is a contributing factor for biliary obstruction.

We previously demonstrated that in Ifnar1-/-Ifngr1-/- or Stat1-/- suckling mice lacking intact type I and type II interferon (IFN) signaling, rhesus rotavirus (RRV) infection causes a lethal disease with clinical manifestations similar to biliary atresia, including acholic stools, oily fur, growth retardation, and excess mortality. Elevated levels of viral RNA are detected in the bile ducts and liver of diseased pups together with severe inflammatory responses in these tissues. However, the viral determinants and the molecular mechanisms driving this process remain incompletely understood. Using an optimized rotavirus (RV) reverse genetics system, we generated a panel of recombinant RVs that encode non-structural protein 1 (NSP1) derived from different RV strains. We found that compared to the parental simian SA11 strain that is less biliary pathogenic, SA11 containing an RRV-derived NSP1 resulted in severe biliary obstructive disease comparable to that associated with RRV infection, reflected by high levels of viral RNA and inflammation in the biliary tract, liver, and pancreas. In contrast, RRV containing an SA11-originated NSP1 showed only mild biliary obstruction comparable to what was observed during SA11 infection. Infection with a monoreassortant RRV virus carrying NSP1 from the bovine RV UK strain also showed substantially reduced viral replication in extra-intestinal organs and did not develop clinical biliary diseases. Mechanistically, RRV NSP1 seemed to promote active viral replication in hepatocytes and this expanded tropism led to enhanced infiltration of CD4 and CD8 T cells, causing immunopathology and damage in the hepatobiliary system. These results highlight an unexpectedly important role of RV NSP1 in viral replication and disease progression in extra-intestinal tissues.

Dead ringer acts as a major regulator of juvenile hormone biosynthesis in insects.

In holometabolous insects, proper control of the production of juvenile hormone (JH), which maintains larval traits, is crucial for successful metamorphosis. JH is produced specifically in the corpora allata (CA) via the functioning of a set of JH biosynthetic enzymes (JHBEs). Expression of JHBE genes in the CA is coordinated except for JH acid methyltransferase (JHAMT), which functions in the last step of JH biosynthesis. Here, we sought to determine the mechanism that enables this coordinated expression, assuming the presence of a central regulator of JHBE genes. Comparison of transcriptomes in the CA during active and inactive stages revealed the presence of 3 transcription factors, whose expression patterns matched those of JHBE genes. We propose that one of these, Dead ringer (Dri), is the central up-regulator of CA-specific JHBE genes including JHAMT, based on the following findings: (ⅰ) Knockdown of Dri in the larvae caused precocious metamorphosis, which was rescued by the exogenous application of JH analog, and (ⅱ) knockdown of Dri decreased the expression of most CA-specific JHBE genes examined. Furthermore, RNAi-based reverse genetics indicated that Dri works most upstream in the control of CA-specific JHBE genes, and that shutdown of JHAMT, which occurs independent of other JHBE genes prior to the onset of metamorphosis, can be hypothetically explained by the presence of an unidentified repressor. Our study suggests that Dri, which has been known to regulate embryonic development in a wide range of animals, is conferred a new role in holometabolous insects, i.e. central regulation of CA-specific JHBE genes.

Development and characterization of reverse genetics systems of feline infectious peritonitis virus for antiviral research

Feline infectious peritonitis (FIP) is a lethal, immune-mediated disease in cats caused by feline infectious peritonitis virus (FIPV), a biotype of feline coronavirus (FCoV). In contrast to feline enteric coronavirus (FECV), which exclusively infects enterocytes and causes diarrhea, FIPV specifically targets macrophages, resulting in the development of FIP. The transmission and infection mechanisms of this complex, invariably fatal disease remain unclear, with no effective vaccines or approved drugs for its prevention or control. In this study, a full-length infectious cDNA clone of the wild-type FIPV WSU79-1149 strain was constructed to generate recombinant FIPV (rFIPV-WT), which exhibited similar growth kinetics and produced infectious virus titres comparable to those of the parental wild-type virus. In addition, the superfold green fluorescent protein (msfGFP) and Renilla luciferase (Rluc) reporter genes were incorporated into the rFIPV-WT cDNA construct to generate reporter rFIPV-msfGFP and rFIPV-Rluc viruses. While the growth characteristics of the rFIPV-msfGFP virus were similar to those of its parental rFIPV-WT, the rFIPV-Rluc virus replicated more slowly, resulting in the formation of smaller plaques than did the rFIPV-WT and rFIPV-msfGFP viruses. In addition, by replacing the S, E, M, and ORF3abc genes with msfGFP and Rluc genes, the replicon systems repFIPV-msfGFP and repFIPV-Rluc were generated on the basis of the cDNA construct of rFIPV-WT. Last, the use of reporter recombinant viruses and replicons in antiviral screening assays demonstrated their high sensitivity for quantifying the antiviral effectiveness of the tested compounds. This integrated system promises to significantly streamline the investigation of virus replication within host cells, enabling efficient screening for anti-FIPV compounds and evaluating emerging drug-resistant mutations within the FIPV genome.

Development of a recombinant non-replicating Newcastle disease virus

ABSTRACT Newcastle disease virus (NDV) is an avian orthoavulavirus I that causes high morbidity and mortality in birds, depending on the pathotype of the virus. Several clinical studies have shown that NDV is a promising oncolytic virus for cancer therapies, yet its application to clinics is restricted due to the pathogenicity of this virus in poultry. Hence, it is important to produce a recombinant NDV (rNDV) with reduced virulence to make it a fit candidate as an oncolytic virus. In this study, we aimed to produce a rNDV with a truncated ant-igenome via reverse genetics to render it non-replicating (nr). The genes responsible for replication were removed from the NDV genome. The rescued nr-NDV was verified, propagated, and purified from tissue culture medium. The virus failed to propagate in embryonated eggs, confirming its non-replicating property. The virus retained its oncolytic activities similar to that of the replicating rAF-GFP as observed in a cell viability assay when a breast cancer cell line, MCF-7, was treated with the virus. In conclusion, this study has successfully generated a system to produce a non-replicating NDV which can be used as a viral vector for heterologous protein delivery, as a safer alternative compared to the virulent strains as an oncolytic virus. RESEARCH HIGHLIGHTS Development of nr-NDV. Reverse transfection was applied for the recovery of nr-NDV. Propagation of nr-NDV was done by sub-passaging transfected BSR T7/5 cells. Safety profile was done to prove that the nr-NDV is non-replicating.

Identification of a cooperative effect between amino acids 169 and 174 in the rotavirus NSP4 double-layered particle-binding domain.

Segmented RNA viruses are capable of exchanging genome segments via reassortment as a means of immune evasion and to maintain viral fitness. Reassortments of single-genome segments are common among group A rotaviruses. Multiple instances of co-reassortment of two genome segments, GS6(VP6) and GS10(NSP4), have been documented in surveillance. Specifically, a division between NSP4 genotypes has been observed in the NSP4 double-layered particle (DLP)-binding domain. A previously hypothesized mechanism for this co-reassortment has been suggested to be the interaction between VP6 and NSP4 during DLP transport from viroplasms for particle maturation. In this study, we used sequence analysis, RNA secondary structure prediction, molecular dynamics and reverse genetics to form a hypothesis regarding the role of the NSP4 DLP-binding domain. Sequence analysis showed that the polarity of NSP4 DLP-binding domain amino acids 169 and 174 is clearly divided between E1 and E2 NSP4 genotypes. Viruses with E1 NSP4s had 169A/I or 169S/T with 174S. E2 NSP4s had 169R/K and 174A. RNA secondary structure prediction showed that mutation in both 545 (aa169) and 561 (aa174) causes global structure remodelling. Molecular dynamics showed that the NSP4/VP6 interaction stability is increased by mutating both aa positions 169 and 174. Using reverse genetics, we showed that an R169I mutation alone does not prevent rescue. Conversely, 174A to 174S prevented rescue, and rescue could be returned by combining 174S with 169I. When compared to rSA11 NSP4-wt, both rSA11 NSP4-R169I and rSA11 NSP4-R169I/A174S had a negligible but significant reduction in titre at specific time points. This study suggests that amino acid 174 of NSP4 may be essential in maintaining the VP6/NSP4 interaction required for DLP transport. Our results suggest that maintenance of specific polarities of amino acids at positions 169 and 174 may be required for the fitness of rotavirus field strains.

Unveiling Novel Insights in Helminth Proteomics: Advancements, Applications, and Implications for Parasitology and Beyond

Helminths have developed intricate mechanisms to survive and evade the host’s immune responses. Hence, understanding the excretory-secretory products (ESPs) by helminths is crucial for developing control tools, including drug targets, vaccines, and potential therapies for inflammatory and metabolic disorders caused by them. Proteomics, the large-scale analysis of proteins, offers a powerful approach to unravel the complex proteomes of helminths and gain insights into their biology. Proteomics, as a science that delves into the functions of proteins, has the potential to revolutionize clinical therapies against parasitic infections that have developed anthelminthic resistance. Proteomic technologies lay a framework for accompanying genomic, reverse genetics, and pharmacokinetic approaches to provide more profound or broader coverage of the cellular mechanisms that underlie the response to anthelmintics. With the development of vaccines against helminth infections, proteomics has brought a major change to parasitology. The proteome of helminths can be analyzed comprehensively, revealing the complex network of proteins that enable parasite survival and pathogenicity. Furthermore, it reveals how parasites interact with hosts’ immune systems. The current article reviews the latest advancements in helminth proteomics and highlights their valuable contributions to the search for anthelminthic vaccines.

Deciphering the evolutionary development of the "Chinese lantern" within Solanaceae.

The key genetic variation underlying the evo-devo of ICS in Solanaceae may be further pinpointed using an integrated strategy of forward and reverse genetics studies under the framework of phylogeny. The calyx of Physalis remains persistent throughout fruit development. Post-flowering, the fruiting calyx is inflated rapidly to encapsulate the berry, giving rise to a "Chinese lantern" structure called inflated calyx syndrome (ICS). It is unclear how this novelty arises. Over the past 2decades, the role of MADS-box genes in the evolutionary development (evo-devo) of ICS has mainly been investigated within Solanaceae. In this review, we analyze the main achievements, challenges, and new progress. ICS acts as a source for fruit development, provides a microenvironment to protect fruit development, and assists in long-distance fruit dispersal. ICS is a typical post-floral trait, and the onset of its development is triggered by specific developmental signals that coincide with fertilization. These signals can be replaced by exogenous gibberellin and cytokinin application. MPF2-like heterotopic expression and MBP21-like loss have been proposed to be two essential evolutionary events for ICS origin, and manipulating the related MADS-box genes has been shown to affect the ICS size, sepal organ identity, and/or male fertility, but not completely disrupt ICS. Therefore, the core genes or key links in the ICS biosynthesis pathways may have undergone secondary mutations during evolution, or they have not yet been pinpointed. Recently, we have made some encouraging progress in acquiring lantern mutants in Physalis floridana. In addition to technological innovation, we propose an integrated strategy to further analyze the evo-devo mechanisms of ICS in Solanaceae using forward and reverse genetics studies under the framework of phylogeny.

Clinical pharmacology functional disorders of narcolepsy in central nervous system

Excessive daytime sleepiness (EDS), cataplexy, and/or other dissociated REM sleep symptoms such hypnagogic hallucinations and sleep paralysis are characteristics of narcolepsy. Antidepressants are being used to treat cataplexy and EDS in narcolepsy patients. These medications have an amphetamine-like effect on the central nervous system (CNS). Other kinds of medications are also used, including gamma-hydroxybutyrate (GHB), a short-acting sedative for EDS/fragmented nighttime sleep, and modafinil, a non-amphetamine wake-promoting drug for EDS. Based on the identification of narcolepsy genes in animals, the primary pathophysiology of human narcolepsy has recently been clarified. The genes responsible for the pathogenesis of canine narcolepsy (hypocretin/orexin ligand and its receptor) have been discovered using both forward (positional cloning) and reverse (mouse gene knockout) genetics. Novel hypothalamic neuropeptides called hypocretins and orexins have a role in a number of hypothalamic processes, including neuroendocrine and energy balance. Although hypocretin-related gene mutations are uncommon in humans, hypocretin-ligand deficiency is a common cause of narcolepsy-cataplexy. The clinical, pathophysiological, and pharmacological facets of narcolepsy are covered in this review.

The greasy finger region of DTMUV NS1 plays an essential role in NS1 secretion and viral proliferation

Duck Tembusu virus (DTMUV) of the Orthoflavivirus genus poses a significant threat to waterfowl aquaculture. Nonstructural protein 1 (NS1), a multifunctional glycoprotein, exists in various oligomeric forms and performs diverse functions. The Greasy Finger (GF) region within NS1 of other flaviviruses has been shown to be a crucial component of the hydrophobic protrusion aiding in anchoring NS1 to the endoplasmic reticulum (ER). However, detailed studies on the role of the GF region in viral proliferation in vitro and the biological properties of NS1 remain scarce. A series of recombinant DTMUV (rDTMUV) with mutations in the GF region, including NS1-F158A, G159A, F160A, G161A, V162A, L163A, F160R, multipoint mutations (GF-4M), or regional deletions (ΔGF), were rescued using a DNA-based reverse genetics system. Only five rDTMUV variants (G159A, F160A, G161A, V162A, and L163A) could be rescued successfully, and these mutations were found to impair replication, reduce virulence, and decrease plaque size, as shown by growth kinetics, duck embryo virulence, and plaque assays, respectively. Upon examining NS1 expression by western blot, we discovered that secreted NS1 (sNS1) presented in large quantities in the supernatant of cells infected with rDTMUV-NS1-G159A, whereas intracellular NS1 was less abundant. These mutations also impacted the primary forms and secretion rates of NS1 in cases of overexpression by western blot and indirect ELISA. Exception for F160A and G161A, which showed decreased secretion rates, all other mutations increased sNS1 expression, with the most pronounced increase observed in F158A and ΔGF, and rDTMUV with these mutations can't be rescued. Co-localization studies of NS1 with the ER demonstrated that the ΔGF mutation attenuated NS1 anchoring to the ER, thereby inhibiting its intracellular residence and promoting secretion. Although these effects vary between flaviviruses, our data reveal that the GF region of NS1 is crucial for viral proliferation and NS1 secretion.

Use of Maize (Zea maysL.) Mutator Transposon-Induced Mutants of theBonnMuResource for Forward and Reverse Genetics Studies

TheBonnMuresource represents a tagged collection of maize (Zea maysL.)Mutator(Mu) transposon-induced mutants, designed for functional genomics studies. Here, we describe the use of theBonnMucollection for identifying and characterizing mutations. Specifically, we describe workflows for use in both reverse and forward genetics strategies in maize. For reverse genetics, users first acquire aBonnMuF2stock of interest based on data accessible at the Maize Genetics and Genomics Database (MaizeGDB). We provide details here for their subsequent propagation and for the confirmation ofMuinsertions by genotyping via PCR, with the ultimate goal of establishing genotype–phenotype relationships of interest. For forward genetics studies, we describe a workflow that involves a combined approach of Mutant-Seq (Mu-Seq) and bulked segregant RNA-seq (BSR-Seq), to identify the causal gene underlying a mutant phenotype of interest.

BonnMu: A Resource for Functional Genomics in Maize (Zea mays L.).

The BonnMu resource is a public transposon-tagged population designed for reverse and forward genetics studies in maize (Zea mays L.). The resource was created by crossing an active Mutator (Mu) transposon line into different inbred lines to induce insertional mutations. The resulting F1 generation was self-pollinated to generate segregating BonnMu F2 stocks. The Mu-tagged BonnMu F2 stocks have insertions in 83% of all annotated maize gene models, and Mu insertion positions and photos of the seedling phenotypes of the segregating BonnMu F2 stocks are deposited in the Maize Genetics and Genomics Database (MaizeGDB), with seeds available to the community. Here, we discuss the creation, expansion, and application of the BonnMu resource for identifying and characterizing mutations induced by Mu transposons, which represents a useful tool for functional genomics studies in maize.

Establishment of a reverse genetics system for virulent systemic feline calicivirus using circular polymerase extension reaction

SummaryFeline caliciviruses can cause oral and upper respiratory tract infections in cats. However, a virulent and systemic feline calicivirus (VS-FCV) variant implicated in multisystem lesions and death in cats has emerged recently. To date, the mechanism underlying virulence variations in VS-FCV remains unclear. The aim of the present study was to provide a tool for exploring genetic variation in VS-FCV, by constructing an infectious clone of VS-FCV SH/2014. First, a full-length cDNA molecular clone of VS-FCV SH/2014 strain, which contains an Xba I recognition site generated by mutating one base (A→T) as a genetic marker, was constructed using the circular polymerase extension reaction (CPER) method. Second, the full-length cDNA clone was introduced into Crandell-Rees feline kidney cells using liposomes to rescue recombinant VS-FCV SH/2014 (rVS-FCV SH/2014). Third, the rescued viruses were identified by real-time PCR, immunofluorescence assay, western blotting, and electron microscopy. The full-length cDNA molecular clone of the VS-FCV SH/2014 strain was successfully constructed and that rVS-FCV SH/2014 could be rescued efficiently. rVS-FCV SH/2014 had the expected genetic markers and morphology and growth characteristics similar to those of the parental virus. The reverse genetics system provides a research platform for future studies on VS-FCV genetic variation and pathogenesis.

Development and Characterization of a New TILLING Population for Forward and Reverse Genetics in Barley (Hordeum vulgare L.).

Mutagenesis is an important tool in crop improvement and free of the regulatory restrictions imposed on genetically modified organisms. Barley (Hordeum vulgare L.) is a diploid species with a genome smaller than those of other members of the Triticeae crops, making it an attractive model for genetic studies in Triticeae crops. In this study, we report an ethyl methane sulfonate (EMS)-mutagenized population in the Chinese barley landrace TX9425, which is tolerant to both abiotic and biotic stress. A TILLING (Targeting Induced Locus Lesion in Genomes) population consisting of 2000 M2 lines was also constructed based on the CEL I enzyme with subsequent polyacrylamide electrophoresis, which decreased the cost and labor investment. The mutant phenotypes of the M2 and M3 generations were scored and revealed the presence of a wide spectrum of morphological diversity. The population was evaluated by screening for induced mutations in five genes of interest. A detailed analysis was performed for the HvGLR3.5 gene and three mutations were identified by screening in 2000 M2 lines. Two of three mutations displayed tuft and yellow striped leaves compared to the wild type. Altogether, our study shows the efficiency of screening and the great potential of the new TILLING population for genetic studies in the barley crop model system.

CryptKeeper: a negative design tool for reducing unintentional gene expression in bacteria.

Foundational techniques in molecular biology-such as cloning genes, tagging biomolecules for purification or identification, and overexpressing recombinant proteins-rely on introducing non-native or synthetic DNA sequences into organisms. These sequences may be recognized by the transcription and translation machinery in their new context in unintended ways. The cryptic gene expression that sometimes results has been shown to produce genetic instability and mask experimental signals. Computational tools have been developed to predict individual types of gene expression elements, but it can be difficult for researchers to contextualize their collective output. Here, we introduce CryptKeeper, a software pipeline that visualizes predictions of bacterial gene expression signals and estimates the translational burden possible from a DNA sequence. We investigate several published examples where cryptic gene expression in E. coli interfered with experiments. CryptKeeper accurately postdicts unwanted gene expression from both eukaryotic virus infectious clones and individual proteins that led to genetic instability. It also identifies off-target gene expression elements that resulted in truncations that confounded protein purification. Incorporating negative design using CryptKeeper into reverse genetics and synthetic biology workflows can help to mitigate cloning challenges and avoid unexplained failures and complications that arise from unintentional gene expression.