Infectious Subgenomic Amplicons method to expedite reverse genetics of SARS-CoV-2 and other coronaviruses

There is a need for simple reverse genetics methods to decipher the biological properties of animal and human coronaviruses. Here, we attempted to rescue the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the Feline enteric coronavirus (FeCoV) using the rapid “Infectious-Subgenomic Amplicons” (ISA) method. For each virus, transfection into permissive cells of eight overlapping subgenomic cDNA fragments covering the entire genome allowed reconstruction of the complete virus genome and generated infectious viral particles. Rescued viruses replicated the phenotypic and genotypic characteristics of the original isolates. In conclusion, the ISA method, which had been previously used for RNA viruses with shorter genomes (e.g., flaviviruses, alphaviruses and enteroviruses) can be used to rescue viruses with substantially longer genomes and usefully complements pre-existing methods for reverse genetics of coronaviruses. Its extreme simplicity and versatility makes it a strong option to decipher the biological properties of coronaviruses circulating in human, domestic or wild fauna populations.

Tobamoviruses of two new species trigger resistance in pepper plants harbouring functional L alleles

Tobamoviruses are often referred to as the most notorious viral pathogens of pepper crops. These viruses are not transmitted by invertebrate vectors, but rather by physical contact and seeds. In this study, pepper plants displaying mild mottle and mosaic symptoms were sampled in four different regions of Peru. Upon double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) tests, seven samples cross-reacted weakly with antibodies against pepper mild mottle virus (PMMoV), suggesting the presence of tobamoviruses. When employing RT-PCR, conserved primers amplified cDNA fragments of viruses from two putative new tobamovirus species in the samples. The complete genome of two representative isolates were, therefore, sequenced and analysed in silico. These viruses, which were tentatively named yellow pepper mild mottle virus (YPMMoV) and chilli pepper mild mottle virus (CPMMoV), shared highest nucleotide genome sequence identities of 83 and 85 % with bell pepper mottle virus (BpeMV), respectively. Mechanical inoculation of indicator plants with YPMMoV and CPMMoV isolates did not show any obvious differences in host ranges. These viruses were also inoculated mechanically on pepper plants harbouring different resistance L alleles to determine their pathotypes. Pepper plants carrying unfunctional L alleles (L 0) to tobamoviruses were infected by all isolates and presented differential symptomatology for YPMMoV and CPMMoV. On the other hand, pepper plants carrying L 1, L 2, L 3 and L 4 alleles were resistant to all isolates, indicating that these viruses belong to pathotype P0.

Rare VPS35 A320V Variant in Taiwanese Parkinson’s Disease Indicates Disrupted CI-MPR Sorting and Impaired Mitochondrial Morphology

Sequence variants in vacuolar protein sorting 35 (VPS35) have been reported to be associated with Parkinson’s disease (PD). To investigate if the genetic variants in VPS35 contribute to Taiwanese PD, VPS35 cDNA fragments from 62 patients with PD were sequenced. A cohort of PD (n = 560) and ethnically matched controls (n = 506) were further examined for the identified mutation. The effects of the mutation on cation-independent mannose-6-phosphate receptor (CI-MPR) sorting and mitochondrial morphology were further examined in 293T cells expressing the mutant VPS35. Here, a novel heterozygous A320V in the VPS35 gene was identified in two late-onset PD (LOPD) patients, which was absent in 506 normal controls. Expression of the A320V mutant in 293T cells demonstrated increased colocalization of VPS35 with CI-MPR and decreased CI-MPR and lysosomal-associated membrane protein 2 (LAMP2) levels. Decreased CI-MPR manifested in missorting of cathepsin D and decreased proteolysis of α-synuclein. A320V mutation also increased mitochondrial E3 ubiquitin protein ligase 1 (MUL1) and thus led to mitofusin 2 (MFN2) degradation. The results suggest that the expression of VPS35 A320V leads to disrupted CI-MPR sorting and impaired mitochondrial morphology, which may partly explain its action in PD.

Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). While the development of specific treatments and a vaccine is urgently needed, functional analyses of SARS-CoV-2 have been limited by the lack of convenient mutagenesis methods. In this study, we established a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. Notably, the construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (~5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. We hope that our reverse genetics system will contribute to the further understanding of SARS-CoV-2.

Infectious Subgenomic Amplicons method to expedite reverse genetics of SARS-CoV-2 and other coronaviruses

There is a need for simple reverse genetics methods to decipher the biological properties of animal and human coronaviruses. Here, we attempted to rescue the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the Feline enteric coronavirus (FeCoV) using the rapid “Infectious-Subgenomic Amplicons” (ISA) method. For each virus, transfection into permissive cells of eight overlapping subgenomic cDNA fragments covering the entire genome allowed reconstruction of the complete virus genome and generated infectious viral particles. Rescued viruses replicated the phenotypic and genotypic characteristics of the original isolates. In conclusion, the ISA method, which had been previously used for RNA viruses with shorter genomes (e.g., flaviviruses, alphaviruses and enteroviruses) can be used to rescue viruses with substantially longer genomes and usefully complements pre-existing methods for reverse genetics of coronaviruses. Its extreme simplicity and versatility makes it a strong option to decipher the biological properties of coronaviruses circulating in human, domestic or wild fauna populations.

cDNA Actin Isolated From Pandanus Sp.

Actin is one of the reference genes that is often used as an internal control in gene expression analysis. This study aimed to isolate actin cDNA from Pandanus sp originated from Riau. Fresh leaves Pandanus sp. Lake Kajuik, Langgam District, Pelalawan Regency, Riau Province. Isolation of RNA, synthesis of total cDNA, amplification of actin genes used McDowell's designed degenerate primer (PlAc46S-20/PlAc245N-20), electrophoresis, sequencing, and data analysis. Actin cDNA fragments obtained were 353 pb in size, registered at GenBank and encoded 117 amino acids. Actin cDNA fragment consists of two exons and one introne. Specific actin primers from Riau Pandanus sp. can be designed based on sequences obtained for the purpose of analyzing certain gene expressions.

Isolation of differentially expressed genes involved in clubroot disease

The interaction between Plasmodiophora brassicae and its host Brassica rapa is investigated by two strategies. (1) IAA-conjugate hydrolases: Root hypertrophy in club root disease is dependent on increased auxin levels and these could result from auxin-conjugate hydrolysis. So far we isolated 5 different cDNA fragments out of various tissues which revealed high identity to IAR3/ILL5, ILL2, ILL3, ILL6 and ILR1 genes from Arabidopsis by comparison with database entries. (2) Random priming: Using this method, we have so far obtained 26 clones from clubroot tissue, from which several sequences may be components of plant signal transduction chains, metabolic pathways and transcriptional regulation.

Identification, expression analysis, and the regulating function on C/EBPs of KLF10 in Dalian purple sea urchin, Strongylocentrotus nudus

Accumulating evidence indicates that Krüppel-like factors (KLFs) play important roles in fat biology via the regulation of CCAAT/enhancer binding proteins (C/EBPs). However, KLFs and C/EBPs have not been identified from Strongylocentrotus nudus, and their roles in this species are not clear. In this study, the full-length cDNA of S. nudus KLF10 (SnKLF10) and three cDNA fragments of S. nudus C/EBPs (SnC/EBPs) were obtained. Examination of tissue distribution and expression patterns during gonadal development implied that SnKLF10 and SnC/EBPs play important roles in gonadal lipogenesis. The presence of transcription factor-binding sites (TFBSs) for KLFs in SnC/EBPs, and the results of an over-expression assay, revealed that SnKLF10 negatively regulates the transcription of SnC/EBPs. In addition, the core promoter regions of SnC/EBPs were determined, and multiple TFBSs for transcription factor (TFs) were identified, which are potential regulators of SnC/EBP transcription. Taken together, these results suggest that SnC/EBP genes are potential targets of SnKLF10, and that SnKLF10 plays a role in lipogenesis by repressing the transcription of SnC/EBPs. These findings provide information for further studies of KLF10 in invertebrates and provide new insight into the regulatory mechanisms of C/EBP transcription.

PASP — a whole-transcriptome poly(A) tail length determination assay for the Illumina platform

The poly(A) tail, co-transcriptionally added to most eukaryotic RNAs, plays an important role in post-transcriptional regulation through modulating mRNA stability and translational efficiency. The length of the poly(A) tail is dynamic, decreasing or increasing in response to various stimuli through the action of enzymatic complexes, and changes in tail length are exploited in regulatory pathways implicated in various biological processes.To date, assessment of poly(A) tail length has mostly relied on protocols targeting only a few transcripts. We present PASP (‘poly(A) tailsequencingprotocol’), a whole-transcriptome approach to measure tail lengths — including a computational pipeline implementing all necessary analyses. PASP uses direct Illumina sequencing of cDNA fragments obtained through G-tailing of poly(A)-selected mRNA followed by fragmentation and reverse transcription.Analysis of reads corresponding to spike-in poly(A) tracts of known length indicated that mean tail lengths can be confidently measured, given sufficient coverage. We further explored the utility of our approach by comparing tail lengths estimated from wild type and Δccr4-1/pan2mutant yeasts. The yeast whole-transcriptome tail length distributions showed high consistency between biological replicates, and the expected upward shift in tail lengths in the mutant samples was detected. This suggests that PASP is suitable for the assessment of global polyadenylation status in yeast.The correlation of per-transcript mean tail lengths between biological and technical replicates was low (higher between mutant samples). Both, however, reached high values after filtering for transcripts with greater coverage. We also compare our results with those of other methods. We identify a number of improvements that could be used in future PASP experiments and, based on our results, believe that direct sequencing of poly(A) tails can become the method of choice for studying polyadenylation using the Illumina platform