Application of CRISPR-Cas12a Enhanced Fluorescence Assay Coupled with Nucleic Acid Amplification for the Sensitive Detection of African Swine Fever Virus.

African swine fever (ASF) is one of the most severe diseases of pigs. In this study, a CRISPR-Cas12a (also known as Cpf1) system coupled with nucleic acid amplification was optimized for the detection of ASF virus (ASFV). Two novel single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters were developed to increase the brightness of the fluorescent signal for the visualization of nucleic acid detection. The CRISPR-Cas12a system was used to simultaneously cleave the polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP) amplicons and the newly developed ssDNA-FQ reporter, resulting in fluorescence that could be easily detected in multiple platforms, especially on cheap and portable blue or UV light transilluminators. This specific cleavage with fluorescence reveals the presence of the amplicon and confirms its identity, thereby preventing false-positive test results from nonspecific amplicons. This method is also uninterfered by the presence of large amounts of irrelevant background DNA and displays no cross-reactivity with other porcine DNA or RNA viruses. When coupled with LAMP, the Cas12a platform can detect a plasmid containing p72 with as few as 2 copies/µL reaction. Our results indicate that the CRISPR-Cas12a enhanced fluorescence assay coupled with nucleic acid amplification is robust, convenient, specific, confirmatory, affordable, and potentially adaptable for ASF diagnosis.

Gene expression profiling of porcine skeletal muscle satellite cells after poly(I:C) stimulation.

Porcine satellite cells (PSCs) play a vital role in the construction, development and self-renewal of skeletal muscle. In this study, PSCs were exposed to poly(I:C) stimulation to mimic viral infection during the proliferation and differentiation phases at 0, 12, 24 and 48 hours (h) of the stimulation. The untreated and treated PSCs were analyzed by the RNA-Seq technology. There were 88, 119, 104 and 95 genes being differentially expressed in 0 h vs 12 h treated, 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated comparison libraries, respectively. The GO terms analysis results showed that during the proliferation phase of treated PSCs, the up-regulated genes related to the immune system were highly expressed. In addition, the gene expressions associated with muscle structure development in response to growth factor emerged during the differentiation phase of untreated PSCs. The biological pathways associated with Influenza A, Toll-like receptor and chemokine signaling were revealed in PSCs following poly(I:C) stimulation. The differentially expressed genes were confirmed by quantitative real-time PCR. These findings expanded our understanding of gene expressions and signaling pathways about the infiltrated mechanism of the virus into PSCs.

Transcriptional Profiling of Leucocyte Count Variation from Porcine Peripheral Blood Reveals Differential Gene Expression.

Leucocytes have tremendous health-check importance related to the individual antiviral capacity of pigs and other mammals. However, the molecular mechanism of the immune response of blood leucocytes in pigs is not completely known. This study investigated the leucocyte-count variation before and after poly I:C stimulation in a Duroc-Erhualian F2 population. Pigs with increased and decreased differences in leucocyte counts were coded as increased responder (IR) and decreased responder (DR), respectively. Then, we used microarray technology to compare the gene-expression profiles of both groups of pigs. Transcriptomic analysis identified 129 differentially expressed genes (DEGs) in IR pigs and 136 DEGs in DR pigs. Forty-one common DEGs showed that both groups had similar expression patterns of immune responses. These results illustrated a differential expression in both groups. Furthermore, qPCR experiment was performed to verify the differential-expression profile. Functional annotation of the DEGs indicated that both IR and DR pigs were similar in several biological processes, including innate immune response, and also exhibited distinct differences in biological processes, molecular function, and pathways. These results provided insights into the mechanism underlying the antiviral capacity of pigs. Trial registration number is CAS Registry Number 24939-03-5.

Selective constraints in cold-region wild boars may defuse the effects of small effective population size on molecular evolution of mitogenomes.

Spatial range expansion during population colonization is characterized by demographic events that may have significant effects on the efficiency of natural selection. Population genetics suggests that genetic drift brought by small effective population size (Ne) may undermine the efficiency of selection, leading to a faster accumulation of nonsynonymous mutations. However, it is still unknown whether this effect might be balanced or even reversed by strong selective constraints. Here, we used wild boars and local domestic pigs from tropical (Vietnam) and subarctic region (Siberia) as animal model to evaluate the effects of functional constraints and genetic drift on shaping molecular evolution. The likelihood-ratio test revealed that Siberian clade evolved significantly different from Vietnamese clades. Different datasets consistently showed that Siberian wild boars had lower Ka/Ks ratios than Vietnamese samples. The potential role of positive selection for branches with higher Ka/Ks was evaluated using branch-site model comparison. No signal of positive selection was found for the higher Ka/Ks in Vietnamese clades, suggesting the interclade difference was mainly due to the reduction in Ka/Ks for Siberian samples. This conclusion was further confirmed by the result from a larger sample size, among which wild boars from northern Asia (subarctic and nearby region) had lower Ka/Ks than those from southern Asia (temperate and tropical region). The lower Ka/Ks might be due to either stronger functional constraints, which prevent nonsynonymous mutations from accumulating in subarctic wild boars, or larger Ne in Siberian wild boars, which can boost the efficacy of purifying selection to remove functional mutations. The latter possibility was further ruled out by the Bayesian skyline plot analysis, which revealed that historical Ne of Siberian wild boars was smaller than that of Vietnamese wild boars. Altogether, these results suggest stronger functional constraints acting on mitogenomes of subarctic wild boars, which may provide new insights into their local adaptation of cold resistance.