Infection Efficiency
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Federica Bove ◽  
Serge Savary ◽  
Laetitia Willocquet ◽  
Vittorio Rossi

AbstractThe cultivation of grape varieties with partial resistance to disease may become an important component for disease management in the future. The impact of partial resistance on downy mildew epidemics according to its components have not been explored so far. This work aims to model, understand, and quantitatively analyse the effect of partial resistance against dual disease epidemics (foliage and clusters) caused by Plasmopara viticola, and rank the efficiency of different resistance components in disease suppression. We use an epidemiological simulation model to integrate the effect of four components of partial resistance, expressed as relative resistance parameters, i.e. infection efficiency (RRIE), latency period (RRLP), sporulation (RRSP), and infectious period (RRIP). Both the individual and combined effect of these components of resistance on downy mildew epidemics are evaluated through a sensitivity analysis. A comparison of simulation runs in different scenarios of disease conduciveness using experimental measurements of components of partial resistance for 16 different grapevine varieties is also performed. Increasing values of RR parameters led to a suppression of disease progress on foliage. The strongest reduction of epidemics on foliage is generated by increases in RRIE, followed by RRSP, RRIP, and last by RRLP. The effect of partial resistance on epidemics is more conspicuous in a scenario of limited disease conduciveness. The strongest suppressive effect of simulated epidemics on clusters is associated with RRIE, and the lowest effect with RRLP, with similar effects of increasing values of RRIP and RRSP. The use of experimentally measured relative resistance parameters to run simulated epidemics shows a reduction of the area under the disease progress curve from 4 × 105 (on a susceptible reference grapevine variety) to 4 × 102 (on cv. Bronner), i.e. a reduction of disease by 1000. The simulation of the varietal effect in intermediate and less favourable scenarios of disease conduciveness strongly suppresses the epidemic on foliage and limits disease on clusters to very low levels. Deploying partial host plant resistance in environments that are not strongly conducive to downy mildew epidemics could represent an effective use of partial resistance.

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1636
A. Katrin Helfer-Hungerbuehler ◽  
Jimit Shah ◽  
Theres Meili ◽  
Eva Boenzli ◽  
Pengfei Li ◽  

Feline leukemia virus (FeLV) is a retrovirus of cats worldwide. High viral loads are associated with progressive infection and the death of the host, due to FeLV-associated disease. In contrast, low viral loads, an effective immune response, and a better clinical outcome can be observed in cats with regressive infection. We hypothesize that by lowering viral loads in progressively infected cats, using CRISPR/SaCas9-assisted gene therapy, the cat’s immune system may be permitted to direct the infection towards a regressive outcome. In a step towards this goal, the present study evaluates different adeno-associated vectors (AAVs) for their competence in delivering a gene editing system into feline cells, followed by investigations of the CRISPR/SaCas9 targeting efficiency for different sites within the FeLV provirus. Nine natural AAV serotypes, two AAV hybrid strains, and Anc80L65, an in silico predicted AAV ancestor, were tested for their potential to infect different feline cell lines and feline primary cells. AAV-DJ revealed superior infection efficiency and was thus employed in subsequent transduction experiments. The introduction of double-strand breaks, using the CRISPR/SaCas9 system targeting 12 selected FeLV provirus sites, was confirmed by T7 endonuclease 1 (T7E1), as well as Tracking of Indels by Decomposition (TIDE) analysis. The highest percentage (up to 80%) of nonhomologous end-joining (NHEJ) was found in the highly conserved gag and pol regions. Subsequent transduction experiments, using AAV-DJ, confirmed indel formation and showed a significant reduction in FeLV p27 antigen for some targets. The targeting of the FeLV provirus was efficient when using the CRISPR/SaCas9 approach in vitro. Whether the observed extent of provirus targeting will be sufficient to provide progressively FeLV-infected cats with the means to overcome the infection needs to be further investigated in vivo.

2021 ◽  
Vol 9 (8) ◽  
pp. 1735
Juan Wan ◽  
Zebao Dai ◽  
Keqin Zhang ◽  
Guohong Li ◽  
Peiji Zhao

Plant parasitic nematodes cause severe damage to crops. Endoparasitic nematophagous fungi (ENF) are a type of important biocontrol fungi, which can cause disease or kill nematodes by producing various spores. As a major ENF, Drechmeria coniospora displays certain potential for controlling plant-parasitic nematodes. In this study, the pathogenicity and secondary metabolites of the endoparasitic fungus D. coniospora YMF1.01759 were investigated. The strain D. coniospora YMF1.01759 had high infection efficiency against nematodes. The process of infecting nematodes by the strain was observed under an electron microscope. Here, 13 metabolites including one new compound 4(S)-butoxy-3-(butoxymethyl)-2-hydroxycyclopent-2-en-1-one (2) were isolated and identified from the fermentation products of D. coniospora YMF1.01759 cultured in a SDAY solid medium. Furthermore, a bioassay showed that 5-hydroxymethylfuran-2-carboxylic acid (1) is toxic to the root knot nematode Meloidogyne incognita and affects the hatching of its egg. Thereby, the nematicidal mortality attained 81.50% at 100 μg/mL for 48 h. Furthermore, egg hatching was inhibited at the tested concentrations, compared with water control eggs. This is the first report on the secondary metabolites of the ENF D. coniospora. The results indicated that D. coniospora could infect nematodes by spores and produce active metabolites to kill nematodes. The biological control potential of D. coniospora against nematodes was expounded further.

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1574
Julia E. Hölper ◽  
Finn Grey ◽  
John Kenneth Baillie ◽  
Tim Regan ◽  
Nicholas J. Parkinson ◽  

Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gD–Pass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gD–Pass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gD–Pass in rabbit RK13-sgms1KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gD–Pass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked.

Wenyang Zhou ◽  
Chang Xu ◽  
Pingping Wang ◽  
Meng Luo ◽  
Zhaochun Xu ◽  

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing an outbreak of coronavirus disease 2019 (COVID-19), has been undergoing various mutations. The analysis of the structural and energetic effects of mutations on protein-protein interactions between the receptor binding domain (RBD) of SARS-CoV-2 and angiotensin converting enzyme 2 (ACE2) or neutralizing monoclonal antibodies will be beneficial for epidemic surveillance, diagnosis, and optimization of neutralizing agents. According to the molecular dynamics simulation, a key mutation N439K in the SARS-CoV-2 RBD region created a new salt bridge with Glu329 of hACE2, which resulted in greater electrostatic complementarity, and created a weak salt bridge with Asp442 of RBD. Furthermore, the N439K-mutated RBD bound hACE2 with a higher affinity than wild-type, which may lead to more infectious. In addition, the N439K-mutated RBD was markedly resistant to the SARS-CoV-2 neutralizing antibody REGN10987, which may lead to the failure of neutralization. The results show consistent with the previous experimental conclusion and clarify the structural mechanism under affinity changes. Our methods will offer guidance on the assessment of the infection efficiency and antigenicity effect of continuing mutations in SARS-CoV-2.

2021 ◽  
Jin-Kui Yang ◽  
Miao-Miao Zhao ◽  
Yun Zhu ◽  
Li Zhang ◽  
Gongxun Zhong ◽  

Abstract The spike (S) protein of SARS coronavirus 2 (SARS-CoV-2) is an ideal target for the development of specific vaccines or drugs. However, treatments targeting viruses with mutant S proteins that have recently emerged in many countries are limited. Cleavage of the S protein by host proteases is essential for viral infection. Here, we discovered two novel sites (CS-1 and CS-2) in the S protein for cleavage by the protease Cathepsin L (CTSL). Both sites are highly conserved among all SARS-CoV-2 variants of concern. Cryo-electron microscopy structural studies revealed that CTSL cleavage increases the dynamics of the receptor binding domain of S and induces novel conformations. In our pseudovirus (PsV) infection experiment, alteration of the cleavage site significantly reduced the infection efficiency, and CTSL inhibitors markedly inhibited infection with PsVs of both the wild-type and emerged SARS-CoV-2 variants. Furthermore, six highly efficient CTSL inhibitors were found to effectively inhibit live virus infection in human cells in vitro, and two of these were further confirmed to prevent live virus infection in human ACE2 transgenic mice in vivo. Our work suggested that the CTSL cleavage sites in SARS-CoV-2 S are emerging new but effective targets for the development of mutation-resistant vaccines and drugs.

2021 ◽  
Giorgio Medici ◽  
Marianna Tassinari ◽  
Giuseppe Galvani ◽  
Stefano Bastianini ◽  
Laura Gennaccaro ◽  

No therapy is currently available for CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD), a severe neurodevelopmental disorder caused by mutations in the CDKL5 gene. Although delivery of a wild-type copy of the mutated gene to cells represents the most curative approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for brain gene therapy. Herein, we used a secretable TATk-CDKL5 protein to enhance the efficiency of a gene therapy for CDD. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to a higher CDKL5 protein replacement due to secretion and transduction of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle-treated Cdkl5 KO mice or Cdkl5 KO mice treated with the AAVPHP.B_CDKL5 vector, indicating that a gene therapy based on a secretable recombinant TATk-CDKL5 protein is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5.

2021 ◽  
Vol 7 (5) ◽  
Chloe Goldsmith ◽  
Damien Cohen ◽  
Anaëlle Dubois ◽  
Maria Guadalupe Martinez ◽  
Kilian Petitjean ◽  

Hepatitis B virus (HBV) contains a 3.2 kb DNA genome and causes acute and chronic hepatitis. HBV infection is a global health problem, with 350 million chronically infected people at increased risk of developing liver disease and hepatocellular carcinoma (HCC). Methylation of HBV DNA in a CpG context (5mCpG) can alter the expression patterns of viral genes related to infection and cellular transformation. Moreover, it may also provide clues as to why certain infections are cleared or persist with or without progression to cancer. The detection of 5mCpG often requires techniques that damage DNA or introduce bias through a myriad of limitations. Therefore, we developed a method for the detection of 5mCpG on the HBV genome that does not rely on bisulfite conversion or PCR. With Cas9-guided RNPs to specifically target the HBV genome, we enriched in HBV DNA from primary human hepatocytes (PHHs) infected with different HBV genotypes, as well as enriching in HBV from infected patient liver tissue, followed by sequencing with Oxford Nanopore Technologies MinION. Detection of 5mCpG by nanopore sequencing was benchmarked with bisulfite-quantitative methyl-specific qPCR (BS-qMSP). The 5mCpG levels in HBV determined by BS-qMSP and nanopore sequencing were highly correlated. Our nanopore sequencing approach achieved a coverage of ~2000× of HBV depending on infection efficiency, sufficient coverage to perform a de novo assembly and detect small fluctuations in HBV methylation, providing the first de novo assembly of native HBV DNA, as well as the first landscape of 5mCpG from native HBV sequences. Moreover, by capturing entire HBV genomes, we explored the epigenetic heterogeneity of HBV in infected patients and identified four epigenetically distinct clusters based on methylation profiles. This method is a novel approach that enables the enrichment of viral DNA in a mixture of nucleic acid material from different species and will serve as a valuable tool for infectious disease monitoring.

2021 ◽  
Hao Zhang ◽  
HuajunSheng Sheng ◽  
Yu Xing ◽  
Qian Liu ◽  
Zhen Guo ◽  

Abstract The gene editing technology CRISPR/Cas9 is presently applied in several fields. Here, we routinely infected A172 and U251 cells with lentiviral vectors in which aquaporin-8 (AQP8) was knocked down using CRISPR/Cas9. Cryopreservation did not significantly alter the viral infection efficiency, but it affected the expression of AQP8 protein and its mRNA in infected cells compared with non-cryopreserved samples. The expression of AQP8 and its mRNA in cryopreserved infected cells that were recovered did not significantly differ from that in the blank and negative controls. This finding indicated that the lentiviral vector lost viability due to cryopreservation at low temperatures and failed to release the AQP8 gene-targeting guide RNA in the infected cells, or the guide RNA was released, but underwent changes that caused it to malfunction in CRISPR/Cas9-mediated AQP8 gene knockdown. This finding might provide a new direction for the prevention and treatment of viral infections.

2021 ◽  
Zaozao Wang ◽  
Bin Kang ◽  
Qianqian Gao ◽  
Lei Huang ◽  
Yingcong Fan ◽  

Abstract Background Mutated KRAS promotes the activation of mitogen-activated protein kinase (MAPK) pathway and the progression of colorectal cancer (CRC) cells. Aberrant activation of phosphatidylinositol 3‑kinase (PI3K) pathway strongly attenuates the efficacy of MAPK suppression in KRAS-mutated CRC cells. The development of a novel strategy targeting dual-pathway is therefore highly essential for the therapy of KRAS-mutated CRC cells. Methods In this study, a quadruple-depleting system for KRAS, MEK1, PIK3CA, and mammalian target of rapamycin (MTOR) genes based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/SaCas9 was developed. Serotype 5 of adenovirus (ADV5) was used as packaging virus for systemic delivery of the CRISPR system. To enhance infection efficiency and specificity of ADV5 to CRC cells and reduce its non-specific tissue tropism, two engineered proteins, an adaptor and a protector were synthesized and formed an ADV-protein complex (APC) when delivered the quadruple-editing system intravenously in vivo. Results The quadruple-editing significantly inhibited MAPK and PI3K pathways in CRC cells with oncogenic mutations of KRAS and PIK3CA or with KRAS mutation and compensated PI3K activation. Compared with MEK and PI3K/MTOR inhibitors, the quadruple-editing induced more significant survival inhibition on primary CRC cells with oncogenic mutations of KRAS and PIK3CA. The adaptor protein which specifically targeting epithelial cell adhesion molecule (EpCAM) could dramatically enhance infection efficiency of ADV5 to CRC cells. and the protector protein could significantly reduce the off-targeting tropisms in a variety of organs. Moreover, the quadruple-editing intravenously delivered by APC significantly blocked dual-pathway and tumor growth, without influencing normal tissues in cell- and patient-derived xenograft models of KRAS-mutated CRC cells. Conclusions APC-delivered quadruple-editing of MAPK and PI3K pathways effectively and specifically blocked the progression of KRAS-mutated CRC cells.

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