Feline calicivirus (FCV) is a well-known causative pathogen for upper respiratory infection in cats. Its high genetic variability challenges existing molecular diagnostic methods in clinical settings. Thus, we developed two sensitive and visual assays for FCV nucleic acid detection based on RPA reaction and CRISPR-Cas13a trans-cleavage activity. Recombinant plasmid DNA, crRNAs, and RPA primers were designed and prepared, respectively, targeting to FCV ORF1 gene. Besides, purified LwCas13a protein was produced by E.coli prokaryotic expression system. To confirm the validity of FCV-Cas13a assays, seven reaction systems (RSs) with different components were tested, and visual readouts were displayed by lateral flow dipstick (FCV-Cas13a-LFD) and fluorescence detector (FCV-Cas13a-FLUOR), respectively. The established FCV-Cas13a assays were capable of detecting FCV nucleic acid in presetting RSs without cross-reaction with other feline-associated pathogens, and the detection limit was as low as 5.5 copies/μl for both visual methods. Moreover, the positive rate of 56 clinical specimens detected by FCV-Cas13a assays (67.9%, 38/56) was notably higher than that of RT-qPCR (44.6%, 25/56) (p < 0.001), including 13 presumptive positive specimens. Taken together, FCV-Cas13a assays provided reliable and visual diagnostic alternatives for FCV field detection.

Background: Feline herpesvirus type 1 (FHV) and Feline calicivirus (FCV) are the primary co-infecting pathogens that cause upper respiratory tract disease in cats. However, there are currently no visual detection assays available for on-site testing. Here, we develop an ultrasensitive and visual detection method based on dual recombinase polymerase amplification (dRPA) reaction and the hybrid Cas12a/Cas13a trans-cleavage activities in a one-tube reaction system, referred to as one-tube dRPA-Cas12a/Cas13a assay. Results: The recombinant plasmid DNAs, crRNAs, and RPA oligonucleotides targeting the FCV ORF1 gene and FHV-1 TK gene were meticulously prepared. Subsequently, dual RPA reactions were performed followed by screening of essential reaction components for hybrid CRISPR-Cas12a (targeting the FHV-1 TK gene) and CRISPR-Cas13a (targeting the FCV ORF1 gene) trans-cleavage reaction. As a result, we successfully established an ultra-sensitive and visually detectable method for simultaneous detection of FCV and FHV-1 nucleic acids using dRPA and CRISPR/Cas-powered technology in one-tube reaction system. Visual readouts were displayed using either a fluorescence detector (Fluor-based assay) or lateral flow dipsticks (LDF-based assay). As expected, this optimized assay exhibited high specificity towards only FHV-1 and FCV without cross-reactivity with other feline pathogens while achieving accurate detection for both targets with limit of detection at 2.4 × 10^− 1 copies/μL for the FHV-1 TK gene and 5.5 copies/μL for the FCV ORF1 gene, respectively. Furthermore, field detection was conducted using the dRPA-Cas12a/Cas13a assay and the reference real-time PCR methods for 56 clinical samples collected from cats with URTD. Comparatively, the results of Fluor-based assay were in exceptional concordance with the reference real-time PCR methods, resulting in high sensitivity (100% for both FHV-1 and FCV), specificity (100% for both FHV-1 and FCV), as well as consistency (Kappa values were 1.00 for FHV-1 and FCV). However, several discordant results for FHV-1 detection were observed by LDF-based assay, which suggests its prudent use and interpretaion for clinical detection. In spite of this, incorporating dRPA-Cas12a/Cas13a assay and visual readouts will facilitate rapid and accurate detection of FHV-1 and FCV in resource-limited settings. Conclusions: The one-tube dRPA-Cas12a/Cas13a assay enables simultaneously ultrasensitive and visual detection of FHV-1 and FCV with user-friendly modality, providing unparalleled convenience for FHV-1 and FCV co-infection surveillance and decision-making of URTD management.

Purpose: With the evolution of dietary habits, obesity has emerged as a significant global health issue. Numerous studies have demonstrated a close association between obesity and gut microbiota; however, the specific contribution of gut microbiota to varying degrees of obesity remains inadequately understood. Consequently, this study aims to characterize the gut microbiota of individuals across different obesity severity levels. Methods: We conducted a comprehensive characterization of the gut microbiome in Chinese obese patients and a healthy control group through the application of 16S rRNA gene sequencing, supplemented by metagenomic sequencing. The study cohort was stratified into five distinct categories based on body mass index (BMI): healthy, overweight, and obesity grades I, II, and III. Results: In obese populations, the gut microbiome structure shifted significantly, with beneficial genera like Faecalibacterium, Roseburia , and Ruminococcus decreasing, and potentially harmful genera such as Blautia, Collinsella , and Streptococcus increasing. These changes impacted host metabolic pathways, including ribosome synthesis, RNA polymerase activity, and DNA repair. Clinical analyses also revealed strong links between specific genera and metabolic markers like lipid metabolism and insulin resistance. Conclusion: Populations with different obesity traits show unique changes in gut flora. The level of dysbiosis, or imbalance in intestinal microbiota, rises with obesity. These microbial changes are linked to host metabolism, indicating that targeting harmful bacteria and supplementing with beneficial ones from normal-weight populations could effectively reduce obesity.

Rabbit hemorrhagic disease (RHD) is an acute fatal disease caused by the rabbit hemorrhagic disease virus (RHDV). Since the first outbreaks of type 2 RHDV (RHDV2) in April 2020 in China, the persistence of this virus in the rabbit population has caused substantial economic losses in rabbit husbandry. Previous failures in preventing RHDV2 prompted us to further investigate the immune mechanisms underlying the virus’s pathogenicity, particularly concerning the spleen, a vital component of the mononuclear phagocyte system (MPS). For this, a previous RHDV2 isolate, CHN/SC2020, was utilized to challenge naive adult rabbits. Then, the splenic transcriptome was determined by RNA-Seq. This study showed that the infected adult rabbits had 3148 differentially expressed genes (DEGs), which were associated with disease, signal transduction, cellular processes, and cytokine signaling categories. Of these, 100 upregulated DEGs were involved in inflammatory factors such as IL1α, IL-6, and IL-8. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these DEGs were significantly enriched in the cytokine–cytokine receptor interaction signaling pathway, which may play a vital role in CHN/SC2020 infection. At the same time, proinflammatory cytokines and chemokines were significantly increased in the spleen at the late stages of infection. These findings suggested that RHDV2 (CHN/SC2020) might induce dysregulation of the cytokine network and compromise splenic immunity against viral infection, which expanded our understanding of RHDV2 pathogenicity.