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BACKGROUNDHow well mouse models recapitulate the transcriptional profiles seen in humans remains debatable, with both conservation and diversity identified in various settings. The K18-hACE2 mouse model has been widely used for evaluation of new interventions for COVID-19.METHODHerein we use RNA-Seq data and bioinformatics approaches to compare the transcriptional responses in the SARS-CoV-2 infected lungs of K18-hACE2 mice with those seen in humans.RESULTSOverlap in differentially expressed genes was generally poor (≈20-30%), even when multiple studies were combined. The overlap was not substantially improved when a second mouse model was examined wherein hACE was expressed from the mouse ACE2 promoter. In contrast, analyses of immune signatures and inflammatory pathways illustrated highly significant concordances between the species.CONCLUSIONAs immunity and immunopathology are the focus of most studies, these hACE2 transgenic mouse models can thus be viewed as representative and relevant models of COVID-19.

Granzyme A (GzmA) is a serine protease secreted by cytotoxic lymphocytes, with GzmA-/- mouse studies informing our understanding of GzmA’s physiological function. We show herein that GzmA-/- mice have a mixed C57BL/6J and C57BL/6N background and retain the full length Nicotinamide Nucleotide Transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in GzmA-/- mice; however, the presence of Nnt, rather than loss of GzmA, was responsible for this phenotype by constraining lymphocyte infiltration. A new CRISPR active site mutant C57BL/6J GzmAS211A mouse provided the first insights into GzmA’s bioactivity free of background issues, with circulating proteolytically active GzmA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of Bioprojects listing C57BL/6J as the mouse strain, had Nnt sequencing reads inconsistent with a C57BL/6J background. The Nnt issue has clearly complicated our understanding of GzmA and may similarly have influenced studies across a broad range of fields.

Poxvirus systems have been extensively used as vaccine vectors. Herein a RNA-Seq analysis of intramuscular injection sites provided detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. The multiple adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1β, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such injection site vaccinology approaches should inform refinements in poxvirus-based vector design., Author summary Poxvirus vector systems have been widely developed for vaccine applications. Despite considerable progress, so far only one recombinant poxvirus vectored vaccine has to date been licensed for human use, with ongoing efforts seeking to enhance immunogenicity whilst minimizing reactogenicity. The latter two characteristics are often determined by early post-vaccination events at the injection site. We therefore undertook an injection site vaccinology approach to analyzing gene expression at the vaccination site after intramuscular inoculation with a recombinant, multiplication defective, vaccinia-based vaccine. This provided detailed insights into inter alia expression of vector-encoded immunoregulatory genes, as well as host innate and adaptive immune responses. We propose that such injection site vaccinology can inform rational vaccine vector design, and we discuss how the information and approach elucidated herein might be used to improve immunogenicity and limit reactogenicity of poxvirus-based vaccine vector systems.

Colorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The BrafV637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.

SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its utility for in vitro and in vivo SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR-/- and IL-28RA-/- mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development., Author summary SARS-CoV-2 uses the human ACE2 (hACE2) receptor to infect cells, but cannot infect mice because the virus cannot bind mouse ACE2 (mACE2). We use an ACE2-lentivirus system in vitro to identify four key amino acids in mACE2 that explain why SARS-CoV-2 cannot infect mice. hACE2-lentivirus was used to express hACE2 in mouse lungs in vivo, with the inflammatory responses after SARS-CoV-2 infection similar to those seen in human COVID-19. Genetically modified mice were used to show that type I and III interferon signaling is required for the inflammatory responses. We also show that the hACE2-lentivirus mouse model can be used to test vaccines. Overall this paper demonstrates that our hACE2-lentivirus system has multiple applications in SARS-CoV-2 and COVID-19 research.

Getah virus (GETV) is a mosquito-transmitted alphavirus primarily associated with disease in horses and pigs in Asia. GETV was also reported to have been isolated from mosquitoes in Australia in 1961, however, retrieval and sequencing of the original isolates (N544 and N554), illustrated that these viruses were virtually identical to the 1955 GETVMM2021 isolate from Malaysia. K-mer mining of the &gt, 40,000 terabases of sequence data in the Sequence Read Archive followed by BLASTn confirmation identified multiple GETV sequences in biosamples from Asia (often as contaminants), but not in biosamples from Australia. In contrast, sequence reads aligning to the Australian Ross River virus (RRV) were readily identified in Australian biosamples. To explore the serological relationship between GETV and other alphaviruses, an adult wild-type mouse model of GETV was established. High levels of cross-reactivity and cross-protection were evident for convalescent sera from mice infected with GETV or RRV, highlighting the difficulties associated with the interpretation of early serosurveys reporting GETV antibodies in Australian cattle and pigs. The evidence that GETV circulates in Australia is thus not compelling.

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.

Formalin fixation and embedding of clinical tissue samples in paraffin is a common method for archiving biological material. These samples are often well annotated and provide an invaluable resource for research. However, this process of fixation and storage of tissue leads to DNA damage and fragmentation. The use of DNA from formalin fixed, paraffin-embedded (FFPE) tissue to interrogate methylation levels on a genome-wide scale can pose challenges. We compared fresh and matched FFPE tissue DNA samples using the Illumina Infinium HD Human Methylation 450K BeadChip platform with a companion application for repair and "restoration" of DNA from FFPE tissue. Our results showed good correlation between fresh and FFPE sample data. FFPE DNA captured 99% of the CpG sites on the array on average. Significant cancer subgroups based on the CpG island methylator phenotype (CIMP) were clearly distinguished for both fresh and FFPE sample sets with cluster and scaling analysis. The DNA methylation status for the five standard CIMP panel genes which was evaluated for all samples by the MethyLight assay was correctly assigned in both fresh and FFPE samples by the array data. We conclude that the "restoration" method followed by assay on the Infinium HD Human Methylation 450K microarray can produce good quality data for DNA from FFPE samples.

Somatic mutations of the BRAF gene are common in melanomas and nevi but the contribution of polymorphisms in this gene to melanoma or nevus susceptibility remains unclear. An Australian melanoma case-control sample was typed for 16 single nucleotide polymorphisms (SNP) within the BRAF gene, and five SNP in three neighboring genes. The sample comprised 755 melanoma cases from 740 families stratified by family history of melanoma and controls from 635 unselected twin families (2,239 individuals). Ancestry of the cases and controls was recorded, and the twins had undergone skin examination to assess total body nevus count, degree of freckling, and pigmentation phenotype. Genotyping was carried out via primer extension followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. SNP in the BRAF gene were found to be weakly associated with melanoma status but not with development of nevi or freckles. The estimated proportion of attributable risk of melanoma due to variants in BRAF is 1.6%. This study shows that BRAF polymorphisms predispose to melanoma but the causal variant has yet to be determined. The burden of disease associated with this variant is greater than that associated with the major melanoma susceptibility locus CDKN2A, which has an estimated attributable risk of 0.2%.

The length polymorphism repeat (5HTTLPR) in the promoter region of the serotonin transporter gene (SLC6A4, also known as 5HTT) is extensively studied in the context of psychiatric phenotypes, particularly in major depressive disorder. However, investigation of this polymorphism in the context of the current generation of large-scale genome-wide association studies is precluded, as the genotyping technology is limited to single-nucleotide polymorphisms (SNPs). Using genome-wide and 5HTTLPR genotype data from a total of 2823 unrelated individuals, we show that no single SNP is in high linkage disequilibrium (LD) with 5HTTLPR but some two-SNP haplotypes provide reasonable predictors. Hence, two-SNP haplotypes can be used as proxies for 5HTTLPR in genome-wide association studies. Analyses are repeated for sets of SNPs that are included in different genome-wide SNP platforms.

A common single nucleotide polymorphism (SNP) in the 5′ untranslated region (5′UTR) of the epidermal growth factor ( EGF ) gene modulates the level of transcription of this gene and hence is associated with serum levels of EGF. This variant may be associated with melanoma risk, but conflicting findings have been reported. An Australian melanoma case–control sample was typed for the EGF+61A>G transversion (rs4444903). The sample comprised 753 melanoma cases from 738 families stratified by family history of melanoma and 2387 controls from 645 unselected twin families. Ancestry of the cases and controls was recorded, and the twins had undergone skin examination to assess total body nevus count, degree of freckling and pigmentation phenotype. SNP genotyping was carried out via primer extension followed by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The EGF+61 SNP was not found to be significantly associated with melanoma status or with development of nevi or freckles. Among melanoma cases, however, G homozygotes had thicker tumors (p=0.05), in keeping with two previous studies. The EGF polymorphism does not appear to predispose to melanoma or nevus development, but its significant association with tumor thickness implies that it may be a useful marker of prognosis.