Infection by the Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory illness and has a high mortality rate (~35%). The requirement for the virus to be manipulated in a biosafety level three (BSL-3) facility has impeded development of urgently-needed antiviral agents. Here, we established anovel mouse model by inserting human dipeptidyl peptidase 4 (hDPP4) into the Rosa26 locus using CRISPR/Cas9, resulting in global expression of the transgene in a genetically stable mouse line. The mice were highly susceptible to infection by MERS-CoV clinical strain hCoV-EMC, which induced severe diffuse pulmonary disease in the animals, and could also be infected by an optimized pseudotyped MERS-CoV. Administration of the neutralizing monoclonal antibodies, H111-1 and m336, as well as a fusion inhibitor peptide, HR2P-M2, protected mice from challenge with authentic and pseudotyped MERS-CoV. These results confirmed that the hDPP4-knockin mouse is a novel model for studies of MERS-CoV pathogenesis and anti-MERS-CoV antiviral agents in BSL-3 and BSL-2facilities, respectively.

Intensive efforts have been made to develop models of hRSV infection or disease using various animals. However, the limitations such as semi-permissiveness and short duration of infection have impeded their applications in both the pathogenesis of hRSV and therapeutics development. Here, we present a mouse model based on a Rag2 gene knockout using CRISPR/Cas9 technology. Rag2^−/− mice sustained high viral loads upon intranasal inoculation with hRSV. The average peak titer rapidly reached 1 × 10^9.8 copies/g and 1c10⁶ TCID₅₀ in nasal cavity, as well as 1 × 10⁸ copies/g and 1 × 10⁵ TCID₅₀ in the lungs up to 5 weeks. Mild interstitial pneumonia, severe bronchopneumonia, elevated cytokines and NK cells were seen in Rag2^−/− mice. A humanized monoclonal antibody showed strong antiviral activity in this animal model, implying that Rag2^−/− mice that support long-term stable infection are a useful tool for studying the transmission and pathogenesis of human RSV, as well as evaluating therapeutics.

Real-time polymerase chain reaction (PCR) has become an increasingly important technique for gene expression profiling because it can provide insights into complex biological and pathological processes and be used to predict disease or treatment outcomes. Although normalized data are necessary for an accurate estimation of mRNA expression levels, several pieces of evidence suggest that the expression of so-called housekeeping genes is not stable. This study aimed to validate reference genes for the normalization of real-time PCR in an N-methyl-N-nitrosourea (MNU)-induced T-cell lymphoma mouse model. T-cell lymphomas were generated in p53-deficient mice by treatment with 37.5 mg/kg MNU. Thymus and spleen were identified as the primary target organs with the highest incidences of lymphomas. We analyzed the RNA expression levels of eight potential endogenous reference genes (Gapdh, Rn18s, Actb, Hprt, B2M, Rplp0, Gusb, Ctbp1). The expression stabilities of these reference genes were tested at different time points after MNU treatment using geNorm and NormFinder algorithms. A total of 65% of MNU-treated mice developed T-cell lymphomas, with the spleen and thymus as the major target organs. All candidate reference genes were amplified efficiently by quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Gene stability evaluation after MNU treatment and during lymphomagenesis revealed that Ctbp1 and Rplp0 were the most stably expressed genes in the thymus and spleen, respectively. RT-PCR of thymus RNA using two additional sets of primer confirmed that Ctbp1 was the most stable of all the candidate reference genes. We provided suitable endogenous controls for gene expression studies in the T-cell lymphoma model.

Human respiratory syncytial virus (RSV) remains a leading cause of severe lower respiratory tract infections in infants and immunocompromised populations, causing approximately 160,000 annual deaths globally. Despite recent approvals of prefusion F (pre-F) protein-based vaccines (Arexvy, Abrysvo) for older adults and pregnant women, pediatric vaccine development faces unique challenges including enhanced respiratory disease (ERD) risks, maternal antibody interference, and immature infant immune responses. Meanwhile, G protein glycosylation variability and NS1/NS2-mediated interferon suppression remain the outstanding difficulties in structure-based vaccine design. Additionally, current animal models demonstrate notable constraints in virus replication, host susceptibility, immune responses, clinical symptoms, and ERD phenomena. This review synthesizes current obstacles and innovative strategies, highlighting that the selection of multi-antigen strategies, appropriate adjuvants, and the development of more precise preclinical animal models are critical elements that will determine the efficacy and safety of future RSV vaccines.

Background: Metabolic dysfunction–associated steatohepatitis (MASH), the progressive phenotype of metabolic dysfunction–associated steatotic liver disease (MASLD), is well recognized for its increased risk of progressing to cirrhosis and hepatocellular carcinoma (HCC). However, early diagnosis and identification of the MASH patients with a tendency toward malignancy HCC remain a significant challenge. Method: In previous studies, we established a novel conditional inducible HRAS gene expression murine model with normal diet and lifestyle conditions, which exhibited 100% HCC incidence and recapitulated the four major progression stages of MASH to HCC: MASH, fibrosis, cirrhosis, and HCC. Based on this model, we revealed the potential markers of MASH prone to HCC characteristics of malignant MASH through RNA‐Seq and bioinformatics analysis, and further confirmed by pathological biopsy, biochemical tests, inflammatory cytokines measurement, Oil O red staining, and immunohistochemical examination. Results: For MASH, the initial stage of HCC, we observed evidence of hyperlipidemia and insulin resistance in the HRAS mouse model based on blood morphology, biochemical parameters, and insulin tolerance test. Furthermore, the pathological features of MASH were confirmed by the presence of hepatocellular fatty vacuolar changes, lipid droplet accumulation, and inflammation. Then, RNA‐Seq analysis revealed the molecular signatures of malignant MASH and revealed three novel potential markers associated with adverse progression of MASH to HCC: Klk1b4, Alox5 , and Pla2g2e . Conclusion: Based on a stable and novel murine model of MASH prone to HCC, we, for the first time, presented a comprehensive molecular signature and identified novel potential adverse progression markers of MASH prone to malignant HCC, which contributed possibly to early clinical diagnosis and prognostic assessment, even becoming potential therapeutic target.

Background: The precise insertion of large DNA fragments (>3–5 kb) remains one of the key obstacles in establishment of genetically modified murine models. Methods: A 21 kb large DNA fragment containing three tandemly linked copies of the human HRAS gene was inserted into the genome of C57BL/6J mouse, generating a mouse model designated as KI.C57‐ras (or named NF‐hHRAS). Whole‐genome sequencing and Sanger sequencing were utilized to it confirm precise insertion and copy number. The stability of transgene expression among different generations was verified from multiple aspects using by digital PCR, western blot and DNA sequencing. To assess tumor susceptibility in the mouse model, N‐Nitroso‐N‐methylurea (MNU) was administered at a dosage of 75 mg/kg. Histopathological examinations were conducted using hematoxylin and eosin (H&E) staining. Results: The HRAS DNA fragment was inserted into mouse chromosome 15E1 site, locating between 80 623 202 bp and 80 625 020 bp. NF‐hHRAS mice exhibited stable inheritance and displayed consistent phenotypes across individuals. Moreover, this mouse model exhibited a high susceptibility to carcinogens. Upon administration of MNU the earliest mortality onset was earlier than that of wild‐type littermates (day 65 vs. day 78 for male and day 56 vs. day 84 for female). Notably, 100% of the NF‐hHRAS transgenic mice developed tumors, with approximately 84% of male NF‐hHRAS mice exhibiting specific tumor types, such as squamous cell carcinoma or squamous cell papilloma, which was consistent with the previously reported carcinogenic rasH2 mouse model. The types of tumors and the target organs exhibited diversity in NF‐hHRAS mice, while the spontaneous tumor incidence remained low (1/50). Conclusions: The NF‐hHRAS mice demonstrated excellent genetic stability, a reproducible phenotype, and high susceptibility to carcinogens, indicating their potential utility in non‐clinical safety evaluations of drugs as per the S1B guidelines issued by the ICH (The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use).