A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry.

It is unknown whether pangolins, the most trafficked mammals, play a role in the zoonotic transmission of bat coronaviruses. We report the circulation of a novel MERS-like coronavirus in Malayan pangolins, named Manis javanica HKU4-related coronavirus (MjHKU4r-CoV). Among 86 animals, four tested positive by pan-CoV PCR, and seven tested seropositive (11 and 12.8%). Four nearly identical (99.9%) genome sequences were obtained, and one virus was isolated (MjHKU4r-CoV-1). This virus utilizes human dipeptidyl peptidase-4 (hDPP4) as a receptor and host proteases for cell infection, which is enhanced by a furin cleavage site that is absent in all known bat HKU4r-CoVs. The MjHKU4r-CoV-1 spike shows higher binding affinity for hDPP4, and MjHKU4r-CoV-1 has a wider host range than bat HKU4-CoV. MjHKU4r-CoV-1 is infectious and pathogenic in human airways and intestinal organs and in hDPP4-transgenic mice. Our study highlights the importance of pangolins as reservoir hosts of coronaviruses poised for human disease emergence.

Unconventional IFNω-like Genes Dominate the Type I IFN Locus and the Constitutive Antiviral Responses in Bats.

Bats are the natural reservoir hosts of some viruses, some of which may spill over to humans and cause global-scale pandemics. Different from humans, bats may coexist with high pathogenic viruses without showing symptoms of diseases. As one of the most important first defenses, bat type I IFNs (IFN-Is) were thought to play a role during this virus coexistence and thus were studied in recent years. However, there are arguments about whether bats have a contracted genome locus or constitutively expressed IFNs, mainly due to species-specific findings. We hypothesized that because of the lack of pan-bat analysis, the common characteristics of bat IFN-Is have not been revealed yet. In this study, we characterized the IFN-I locus for nine Yangochiroptera bats and three Yinpterochiroptera bats on the basis of their high-quality bat genomes. We also compared the basal expression in six bats and compared the antiviral and antiproliferative activity and the thermostability of representative Rhinolophus bat IFNs. We found a dominance of unconventional IFNω-like responses in the IFN-I system, which is unique to bats. In contrast to IFNα-dominated IFN-I loci in the majority of other mammals, bats generally have shorter IFN-I loci with more unconventional IFNω-like genes (IFNω or related IFNαω), but with fewer or even no IFNα genes. In addition, bats generally have constitutively expressed IFNs, the highest expressed of which is more likely an IFNω-like gene. Likewise, the highly expressed IFNω-like protein also demonstrated the best antiviral activity, antiproliferative activity, or thermostability, as shown in a representative Rhinolophus bat species. Overall, we revealed pan-bat unique, to our knowledge, characteristics in the IFN-I system, which provide insights into our understanding of the innate immunity that contributes to a special coexistence between bats and viruses.

The Zinc Finger Protein MaCCCH33-Like2 Positively Regulates Banana Fruit Ripening by Modulating Genes in Starch and Cell Wall Degradation.

As zinc finger protein transcription factors (TFs), the molecular mechanism of Cys-Cys-Cys-His (CCCH) TFs in regulating plant development, growth and stress response has been well studied. However, the roles of CCCH TFs in fruit ripening are still obscure. Herein, we report that MaCCCH33-like2 TF and its associated proteins modulate the fruit softening of 'Fenjiao' bananas. MaCCCH33-like2 interacts directly with the promoters of three genes: isoamylase2 (MaISA2), sugar transporter14-like (MaSUR14-like) and ß-d-xylosidase23 (MaXYL23), all of which are responsible for encoding proteins involved in the degradation of starch and cell wall components. Additionally, MaCCCH33-like2 forms interactions with abscisic acid-insensitive 5 (ABI5)-like and ethylene F-box protein 1 (MaEBF1), resulting in enhanced binding and activation of promoters of genes related to starch and cell wall degradation. When MaCCCH33-like2 is transiently and ectopically overexpressed in 'Fenjiao' banana and tomato fruit, it facilitates softening and ripening processes by promoting the degradation of cell wall components and starch and the production of ethylene. Conversely, the temporary silencing of MaCCCH33-like2 using virus-induced gene silencing (VIGS) inhibits softening and ripening in the 'Fenjiao' banana by suppressing ethylene synthesis, as well as starch and cell wall degradation. Furthermore, the promoter activity of MaCCCH33-like2 is regulated by MaABI5-like. Taken together, we have uncovered a novel MaCCCH33-like2/MaEBF1/MaABI5-like module that participates in fruit softening regulation in bananas.

N-linked glycoproteins and host proteases are involved in swine acute diarrhea syndrome coronavirus entry.

IMPORTANCE: Gaining insight into the cell-entry mechanisms of swine acute diarrhea syndrome coronavirus (SADS-CoV) is critical for investigating potential cross-species infections. Here, we demonstrated that pretreatment of host cells with tunicamycin decreased SADS-CoV attachment efficiency, indicating that N-linked glycosylation of host cells was involved in SADS-CoV entry. Common N-linked sugars Neu5Gc and Neu5Ac did not interact with the SADS-CoV S1 protein, suggesting that these molecules were not involved in SADS-CoV entry. Additionally, various host proteases participated in SADS-CoV entry into diverse cells with different efficiencies. Our findings suggested that SADS-CoV may exploit multiple pathways to enter cells, providing insights into intervention strategies targeting the cell entry of this virus.

F-box protein EBF1 and transcription factor ABI5-like regulate banana fruit chilling-induced ripening disorder.

Cold stress adversely affects plant production, both qualitatively and quantitatively. Banana (Musa acuminata) is sensitive to cold stress and suffers chilling injury (CI) when stored under 11°C, causing abnormal fruit softening. However, the mechanism underlying the abnormal fruit softening due to CI remains obscure. This study uncovered the coordinated transcriptional mechanism of ethylene F-box (EBF1) protein and abscisic acid-insensitive 5 (ABI5)-like protein in regulating chilling-induced softening disorders of Fenjiao banana. Cold stress severely inhibited the transcript and protein levels of EBF1, ABI5-like, and fruit softening-related genes. The ABI5-like protein bound to the promoters of key starch and cell wall degradation-related genes such as ß-amylase 8 (BAM8), pectate lyase 8 (PL8), and ß-D-xylosidase23-like (XYL23-like) and activated their activities. EBF1 physically interacted with ABI5-like and enhanced the transcriptional activity of the key starch and cell wall degradation-related genes but did not ubiquitinate or degrade ABI5-like protein. This promoted fruit ripening and ameliorated fruit CI in a manner similar to the effect of exogenous abscisic acid treatment. The ectopic and transient overexpression of EBF1 and ABI5-like genes in tomato (Solanum lycopersicum) and Fenjiao banana accelerated fruit ripening and softening by promoting ethylene production, starch and cell wall degradation, and decreasing fruit firmness. EBF1 interacted with EIL4 but did not ubiquitinate or degrade EIL4, which is inconsistent with the typical role of EBF1/2 in Arabidopsis (Arabidopsis thaliana). These results collectively highlight that the interaction of EBF1 and ABI5-like controls starch and cell wall metabolism in banana, which is strongly inhibited by chilling stress, leading to fruit softening and ripening disorder.

Tupaia GBP1 Interacts with STING to Initiate Autophagy and Restrict Herpes Simplex Virus Type 1 Infection.

Stimulator of IFN genes (STING) is a key molecule that binds to cyclic dinucleotides produced by the cyclic GMP-AMP synthase to activate IFN expression and autophagy in the fight against microbial infection. The regulation of STING in the activation of IFN expression has been extensively reported, whereas the regulation of STING in the initiation of autophagy is still insufficiently determined. IFN-inducible guanylate-binding proteins (GBPs) are central to the cell-autonomous immunity in defending a host against viral, bacterial, and protozoan infections. In this study using the Chinese tree shrew (Tupaia belangeri chinensis), which is genetically close to primates, we found that Tupaia GBP1 (tGBP1) combines with Tupaia STING (tSTING), promotes autophagy, and moderately inhibits HSV type 1 (HSV-1) infection. The antiviral effects of tGBP1 are IFN independent. Mechanistically, tGBP1 interacted with tSTING, Tupaia sequestosome 1, and Tupaia microtubule associated protein 1 L chain 3, forming a complex which promotes autophagy in response to HSV-1 infection. This function of tGBP1 against HSV-1 infection was lost in tSTING knockout cells. Overexpression of either tSTING or its mutant tSTING-ΔCTT that can only activate autophagy rescued the anti-HSV-1 activity of tGBP1 in tSTING knockout cells. Our study not only elucidated the underlying mechanism of tGBP1 antiviral activity against HSV-1 infection, but also uncovered the regulation of tSTING in the initiation of autophagy in response to HSV-1 infection.

Maternal exposure to chiral triazole fungicide tebuconazole induces enantioselective thyroid disruption in zebrafish offspring.

Pesticides could induce long-term impacts on aquatic ecosystem via transgenerational toxicity. However, for many chiral pesticides, the potential enantioselectivity of transgenerational toxicity has yet to be fully understood. In this study, we used zebrafish as models to evaluate the maternal transfer risk of tebuconazole (TEB), which is a chiral triazole fungicide currently used worldwide and has been frequently detected in surface waters. After 28-day food exposure (20 and 400 ng/g) to the two enantiomers of TEB (S- and R-TEB) in adult female zebrafish (F0), increased malformation rate and decreased swimming speed were found in F1 larvae, with R-TEB showing higher impacts than S-enantiomer. Additionally, enantioselective effects on the secretion of thyroid hormones (THs) and expression of TH-related key genes along the hypothalamic-pituitary-thyroid (HPT) axis were found in both F0 and F1 after maternal exposure. Both the two enantiomers significantly disrupted the triiodothyronine (T3) and thyroxine (T4) contents in F0 with different degrees, whereas in F1, significant effects were only found in R-TEB groups with decreasing of both T3 and T4 contents. Most of the HPT axis related genes in F0 were upregulated by TEB and more sensitive to R-TEB than to S-TEB. In contrast, most of the genes in F1 were downregulated by both R- and S-TEB, especially the genes that are primarily responsible for thyroid development and growth (Nkx2-1), TH synthesis (NIS and TSHꞵ) and metabolism (Deio1). Findings from this study highlight the key role of enantioselectivity in the ecological risk assessment of chiral pesticides through maternal transfer.

MaBEL1 regulates banana fruit ripening by activating cell wall and starch degradation-related genes.

Banana is a typical subtropical fruit, sensitive to chilling injuries and prone to softening disorder. However, the underlying regulatory mechanisms of the softening disorder caused by cold stress remain obscure. Herein, we found that BEL1-LIKE HOMEODOMAIN transcription factor 1 (MaBEL1) and its associated proteins regulate the fruit softening and ripening process. The transcript and protein levels of MaBEL1 were up-regulated with fruit ripening but severely repressed by the chilling stress. Moreover, the MaBEL1 protein interacted directly with the promoters of the cell wall and starch degradation-related genes, such as MaAMY3, MaXYL32, and MaEXP-A8. The transient overexpression of MaBEL1 alleviated fruit chilling injury and ripening disorder caused by cold stress and promoted fruit softening and ripening of "Fenjiao" banana by inducing ethylene production and starch and cell wall degradation. The accelerated ripening was also validated by the ectopic overexpression in tomatoes. Conversely, MaBEL1-silencing aggravated the chilling injury and ripening disorder and repressed fruit softening and ripening by inhibiting ethylene production and starch and cell wall degradation. MaABI5-like and MaEBF1, the two positive regulators of the fruit softening process, interacted with MaBEL1 to enhance the promoter activity of the starch and cell wall degradation-related genes. Moreover, the F-box protein MaEBF1 does not modulate the degradation of MaBEL1, which regulates the transcription of MaABI5-like protein. Overall, we report a novel MaBEL1-MaEBF1-MaABI5-like complex system that mediates the fruit softening and ripening disorder in "Fenjiao" bananas caused by cold stress.

An Alternative Splicing of Tupaia STING Modulated Anti-RNA Virus Responses by Targeting MDA5-LGP2 and IRF3.

The stimulator of IFN genes (STING; also known as MITA, TMEM173, MPYS, or ERIS) is generally regarded as a key adaptor protein for sensing pathogenic DNA genomes. However, its role in RNA viral signaling as part of the innate immunity system remains controversial. In this study, we identified two isoforms of STING (a full-length Tupaia STING [tSTING-FL] and a Tupaia STING short isoform [tSTING-mini]) in the Chinese tree shrew (Tupaia belangeri chinensis), a close relative of primates. tSTING-FL played a key role in the HSV-1-triggered type I IFN signaling pathway, whereas tSTING-mini was critical for RNA virus-induced antiviral signaling transduction. tSTING-mini, but not tSTING-FL, interacted with tMDA5-tLGP2 and tIRF3 in resting cells. Upon RNA virus infection, tSTING-mini caused a rapid enhancement of the tMDA5-tLGP2-mediated antiviral response and acted earlier than tSTING-FL. Furthermore, tSTING-mini was translocated from the cytoplasm to the nucleus during RNA virus infection and promoted tIRF3 phosphorylation through tSTING-mini-tIRF3 interaction, leading to a restriction of viral replication. After the initiation of antiviral effect, tSTING-mini underwent rapid degradation by tDTX3L-tPAPR9 via k48-linked ubiquitination through a proteasome-dependent pathway. Our results have shown alternative isoforms of STING counteract RNA virus infection in different ways.

Tupaia OASL1 Promotes Cellular Antiviral Immune Responses by Recruiting MDA5 to MAVS.

Melanoma differentiation-associated gene 5 (MDA5) is a key cytoplasmic dsRNA sensor. Upon binding to invading viral RNA, activated MDA5 is recruited to mitochondria and interacts with mitochondrial antiviral signaling gene (MAVS) to initiate innate antiviral immune responses. The elegant regulation of this process remains elusive. In this study, using the Chinese tree shrew (Tupaia belangeri chinensis), which is genetically close to primates, we identified the Tupaia oligoadenylate synthetases-like 1 (tOASL1) as a positive regulator of the Tupaia MDA5 (tMDA5) and Tupaia MAVS (tMAVS)-mediated IFN signaling. Overexpression of tOASL1 significantly potentiated the RNA virus-triggered induction of the type I IFNs and downstream antiviral genes. Conversely, knockdown of tOASL1 had an impaired antiviral immune response. Mechanistically, tOASL1 was associated with mitochondria and directly interacted with tMDA5 and tMAVS. Upon RNA virus infection, tOASL1 enhanced the interaction between tMDA5 and tMAVS via its OAS and UBL domains. Our results revealed a novel mechanism by which tOASL1 contributes to host antiviral responses via enhancing tMDA5 and tMAVS interaction.

Tupaia MAVS Is a Dual Target during Hepatitis C Virus Infection for Innate Immune Evasion and Viral Replication via NF-κB.

Hepatitis C virus (HCV) infection is the cause of severe liver disease in many people. The restricted species tropism of HCV hinders the research and development of drugs and vaccines. The Chinese tree shrew (Tupaia belangeri chinensis) is a close relative of primates and can be infected by HCV, but the underlying mechanisms are unknown. In this study, we have characterized the functions of tree shrew MAVS (tMAVS) in response to HCV infection and defined the capacity of HCV replication. HCV was shown to be colocalized with tMAVS in primary tree shrew hepatocytes and cleaved tMAVS at site Cys508 via its NS3/4A protease, with a modulating effect by site Glu506 of tMAVS. The tMAVS cleavage by HCV NS3/4A impaired the IRF3-mediated induction of IFN-ß but maintained the activated NF-κB signaling in the tree shrew primary cells. Activation of the tMAVS-dependent NF-κB signaling inversely inhibited HCV replication and might limit the establishment of persistent infection. Overall, our study has revealed an elegant example of the balance between the host defenses and HCV infection via the MAVS-mediated antiviral signaling and has provided an insight into the mechanisms underpinning HCV infection in the Chinese tree shrew.

Tupaia guanylate-binding protein 1 interacts with vesicular stomatitis virus phosphoprotein and represses primary transcription of the viral genome.

Chinese tree shrews (Tupaia belangeri chinensis) are increasingly used as an alternative experimental animal to non-human primates in studying viral infections. Guanylate-binding proteins (GBP) belong to interferon (IFN)-inducible GTPases and defend the mammalian cell interior against diverse invasive pathogens. Previously, we identified five tree shrew GBP genes (tGBP1, tGBP2, tGBP4, tGBP5, and tGBP7) and found that tGBP1 showed antiviral activity against vesicular stomatitis virus (VSV) and type 1 herpes simplex virus (HSV-1) infections. Here, we showed that the anti-VSV activity of tGBP1 was independent of its GTPase activity and isoprenylation. In response to VSV infection, instead of regulating IFN expression and autophagy, tGBP1 competed with the VSV nucleocapsid (N) protein in binding to the VSV phosphoprotein (VSV-P), leading to the repression of the primary transcription of the VSV genome. These observations constitute the first report of the potential mechanism underlying the inhibition of VSV by GBP1.

Molecular characterization of the 2',5'-oligoadenylate synthetase family in the Chinese tree shrew (Tupaia belangeri chinensis).

Virus infection induces type I interferons (IFNs) that in turn exert their pleiotropic effects through inducing a large number of interferon-stimulated genes (ISGs). The IFN-induced 2',5'-oligoadenylate synthetases (OASs) have been identified as a member of the ISGs family characterized by the ability to synthesize 2',5'-oligoadenylate (2-5A), which can induce the degradation of viral RNA by activating RNase L within the infected cells to block viral replications. In this study, we characterized the OASs of the Chinese tree shrew (Tupaia belangeri chinensis), a small mammal genetically close to primates and has the potential as animal model for viral infections. We identified 4 putative tree shrew OASs (tOASs, including tOAS1, tOAS2, tOASL1, and tOASL2) and characterized their roles in antiviral responses. Tree shrew lost tOAS3 that was presented in human and mouse. Phylogenetic analyses based on the protein sequences showed a close relationship of tOASs with those of mammals. Constitutive mRNA expression of tOASs was found in seven tissues (heart, liver, spleen, lung, kidney, small intestine and brain). Moreover, tOASs were significantly up-regulated upon various virus infections. Overexpression of tOASs significantly inhibited DNA virus and RNA virus replications in tree shrew primary renal cells. tOAS1 and tOAS2, but not tOASL1 and tOASL2, exerted their anti-HSV activity in an RNase L-dependent pathway. Collectively, our results revealed the evolutionary conservation of tOASs in tree shrew and might offer helpful information for creating viral infection models using the Chinese tree shrew.

Establishment and characterization of an immortalized renal cell line of the Chinese tree shrew (Tupaia belangeri chinesis).

The Chinese tree shrew holds a great potential as a viable animal model in biomedical research, especially for infectious diseases and neuropsychiatric disorders. A thorough understanding of the innate immunity, which represents the first line that defends the host against viral infection, of the Chinese tree shrew, is needed. However, the progress is hindered by the lack of a proper cell line for research usage. In this study, we established a cell line that is applicable to the study of tree shrew innate immune responses against viral infections. The Chinese tree shrew primary renal cells (TSPRCs) were immortalized by simian virus 40 large T antigen (SV40LT) transduction, and the immortalized cells were termed TSR6 (tree shrew renal cell #6). TSR6 showed a similar morphology to TSPRCs and expressed the epithelial cell-specific marker cytokeratin 18 (KRT18). In addition, TSR6 could be transfected by transfection reagent and was suitable for CRISPR/Cas9-mediated gene editing. Infection of Newcastle disease virus (NDV) or herpes simplex virus 1 (HSV-1) in TSR6 induced the mRNA expression of tree shrew interferon-ß (tIFNB1) and myxovirus resistance protein 1 (tMx1) in a dose- and time-dependent manner. Collectively, we successfully established a tree shrew renal cell line and demonstrated that this cell line was suitable for the study of the innate immune response to viral infections.

A Preliminary Analysis of Wind Retrieval, Based on GF-3 Wave Mode Data.

This paper presents an analysis of measurements of the normalized radar cross-(NRCS) in Wave Mode for Chinese C-band Gaofen-3(GF-3) synthetic aperture radar (SAR). Based on 2779 images from GF-3 quad-polarization SAR in Wave Mode and collocated wind vectors from ERA-Interim, this experiment verifies the feasibility of using ocean surface wind fields and VV-polarized NRCS to perform normalized calibration. The method uses well-validated empirical C-band geophysical model function (CMOD4) to estimate the calibration constant for each beam. In addition, the relationship between cross-pol NRCS and wind vectors is discussed. The cross-pol NRCS increases linearly with wind speed and it is obviously modulated by the wind direction when the wind speed is greater than 8 m/s. Furthermore, the properties of the polarization ratio, denoted PR, are also investigated. The PR is dependent on incidence angle and azimuth angle. Two empirical models of the PR are fitted, one as a function of incidence angle only, the other with additional dependence on azimuth angle. Assessments show that the σ VV 0 retrieved from new PR models as well as σ HH 0 is in good agreement with σ VV 0 extracted from SAR images directly.

SARS-CoV-2 NSP14 induces AP-1 transcriptional activity via its interaction with MEK.

The NSP14 protein of SARS-CoV-2 not only facilitates viral replication but also plays a pivotal role in activating the host immune system by enhancing cytokine production. In this study, we found that NSP14 markedly activated the activator protein 1 (AP-1) pathway by increasing the phosphorylation of ERK (p-ERK), which enters the nucleus and promotes AP-1 transcription. The screening of the main proteins of the ERK pathway revealed that NSP14 could interact with MEK, a kinase of ERK, and increase the level of phosphorylated MEK. The addition of the MEK inhibitor U0126 suppressed the level of p-ERK induced by NSP14 and partly blocked cytokine production, suggesting that NSP14 activates MEK to enhance AP-1 signaling. Further investigation demonstrated that the ExoN domain of NSP14 might be crucial for the interaction and activation of MEK. These results suggest a novel mechanism by which NSP14 of SARS-CoV-2 induces a proinflammatory response in the host.

MaC2H2-like regulates chilling stress response of 'Fenjiao' banana by modulating flavonoid synthesis and fatty acid desaturation.

Chilling injury (CI) is a major problem that affects fruit quality and ripening. Herein, chilling stress severely inhibited the expression of transcription factor MaC2H2-like. MaC2H2-like activates the expression of genes associated with flavonoid synthesis (MaC4H-like1, Ma4CL-like1, MaFLS, and MaFLS3) and fatty acid desaturation (MaFAD6-2 and MaFAD6-3), the leading indicators of chilling tolerance. MaC2H2-like interacts with MaEBF1 and boosts the transcriptional activity of MaFAD6-2, MaFAD6-3, Ma4CL-like1, and MaFLS. The overexpression of MaC2H2-like reduced fruit CI, induced the expression of these genes and increased the content of flavonoid and unsaturated fatty acid. Meanwhile, the silencing of MaC2H2-like increased fruit CI and downregulated the expression of those genes and reduced the content of flavonoid and unsaturated fatty acid. These results indicate that MaC2H2-like function as new player in modulating fruit CI by regulating flavonoid synthesis and fatty acid desaturation. MaC2H2-like could be a useful candidate gene for improving cold tolerance in 'Fenjiao' banana.

Ebola virus VP35 perturbs type I interferon signaling to facilitate viral replication.

As one of the deadliest viruses, Ebola virus (EBOV) causes lethal hemorrhagic fevers in humans and nonhuman primates. The suppression of innate immunity leads to robust systemic virus replication of EBOV, leading to enhanced transmission. However, the mechanism of EBOV-host interaction is not fully understood. Here, we identified multiple dysregulated genes in early stage of EBOV infection through transcriptomic analysis, which are highly clustered to Jak-STAT signaling. EBOV VP35 and VP30 were found to inhibit type I interferon (IFN) signaling. Moreover, exogenous expression of VP35 blocks the phosphorylation of endogenous STAT1, and suppresses nuclear translocation of STAT1. Using serial truncated mutations of VP35, N-terminal 1-220 amino acid residues of VP35 were identified to be essential for blocking on type I IFN signaling. Remarkably, VP35 of EBOV suppresses type I IFN signaling more efficiently than those of Bundibugyo virus (BDBV) and Marburg virus (MARV), resulting in stable replication to facilitate the pathogenesis. Altogether, this study enriches understanding on EBOV evasion of innate immune response, and provides insights into the interplay between filoviruses and host.

Calibrate the non-orthogonal error of AFM with two-dimensional self-traceable grating.

The calibration of the non-orthogonal error in nanoscale measurements is of paramount importance for analytical measuring instruments. Particularly, the calibration of non-orthogonal errors in atomic force microscopy (AFM) is essential for the traceable measurements of novel materials and two-dimensional (2D) crystals. The 2D self-traceable grating with a theoretical non-orthogonal angle of less than 0.0027° and an expanded uncertainty of 0.003° (k = 2) are measured by the Metrological Large Range Scanning Probe Microscope (Met. LR-SPM). In this study, we characterized the local and overall non-orthogonal error in AFM scans and proposed a protocol to tune the optimal scanning parameters of AFM minimizing the non-orthogonal error. We presented the method for accurately calibrating a commercial AFM system for non-orthogonal by establishing a detailed uncertainty budget and errors analysis. Our results verified the important advantages of the 2D self-traceable grating in calibrating precision instruments.