Enterovirus G (EV-G) is prevalent in pig populations worldwide, and a total of 20 genotypes (G1 to G20) have been confirmed. Recently, recombinant EV-Gs carrying the papain-like cysteine protease (PLCP) gene of porcine torovirus have been isolated or detected, while their pathogenicity is poorly understood. In this study, an EV-G17-PLCP strain, ‘EV-G/YN23/2022’, was isolated from the feces of pigs with diarrhea, and the virus replicated robustly in numerous cell lines. The isolate showed the highest complete genome nucleotide (87.5%) and polyprotein amino acid (96.6%) identity in relation to the G17 strain ‘IShi-Ya4’ (LC549655), and a possible recombination event was detected at the 708 and 3383 positions in the EV-G/YN23/2022 genome. EV-G/YN23/2022 was nonlethal to piglets, but mild diarrhea, transient fever, typical skin lesions, and weight gain deceleration were observed. The virus replicated efficiently in multiple organs, and the pathological lesions were mainly located in the small intestine. All the challenged piglets showed seroconversion for EV-G/YN23/2022 at 6 to 9 days post-inoculation (dpi), and the neutralization antibody peaked at 15 dpi. The mRNA expression levels of IL-6, IL-18, IFN-α, IFN-β, and ISG-15 in the peripheral blood mononuclear cells (PBMCs) were significantly up-regulated during viral infection. This is the first documentation of the isolation and pathogenicity evaluation of the EV-G17-PLCP strain in China. The results may advance our understanding of the evolution characteristics and pathogenesis of EV-G-PLCP.

Replication of viral genome is the central event during the lytic infectious cycle of herpes simplex virus 1 (HSV-1). However, the details of HSV-1 replication process are still elusive due to the limitations of current molecular and conventional fluorescent microscopy methods. Stimulated emission depletion (STED) microscopy is one of the recently available super-resolution techniques allowing observation at sub-diffraction resolution. To gain new insight into HSV-1 replication, we used a combination of stimulated emission depletion microscopy, fluorescence in situ hybridization (FISH) and immunofluorescence (IF) to observe the HSV-1 replication process. Using two colored probes labeling the same region of HSV-1 genome, the two probes highly correlated in both pre-replication and replicating genomes. In comparison, when probes from different regions were used, the average distance between the two probes increased after the virus enters replication, suggesting that the HSV-1 genome undergoes dynamic structure changes from a compact to a relaxed formation and occupies larger space as it enters replication. Using FISH and IF, viral single strand binding protein ICP8 was seen closely positioned with HSV-1 genome. In contrast, ICP8 and host RNA polymerase II were less related. This result suggests that ICP8 marked regions of DNA replication are spatially separated from regions of active transcription, represented by the elongating form of RNA polymerase II within the viral replication compartments. Comparing HSV-1 genomes at early stage of replication with that in later stage, we also noted overall increases among different values. These results suggest stimulated emission depletion microscopy is capable of investigating events during HSV-1 replication. 1) Replicating HSV-1 genome could be observed by super-resolution microscopy; 2) Viral genome expands spatially during replication; 3) Viral replication and transcription are partitioned into different sub-structures within the replication compartments.

Introduction: bluetongue virus (BTV) infection triggers dramatic and complex changes in the host's transcriptional profile to favor its own survival and reproduction. However, there is no whole-transcriptome study of susceptible animal cells with BTV infection, which impedes the in-depth and systematical understanding of the comprehensive characterization of BTV-host interactome, as well as BTV infection and pathogenic mechanisms.Methods: to systematically understand these changes, we performed whole-transcriptome sequencing in BTV serotype 1 (BTV-1)-infected and mock-infected sheep embryonic testicular cells, and subsequently conducted bioinformatics differential analyses.Results: there were 1504 differentially expressed mRNAs, 78 differentially expressed microRNAs, 872 differentially expressed long non-coding RNAs, and 59 differentially expressed circular RNAs identified in total. Annotation from the Gene Ontology, enrichment from the Kyoto Encyclopedia of Genes and Genomes, and construction of competing endogenous RNA networks revealed differentially expressed RNAs primarily related to virus-sensing and signaling transduction pathways, antiviral and immune responses, inflammation, and development and metabolism related pathways. Furthermore, a protein-protein interaction network analysis found that BTV may contribute to abnormal spermatogenesis by reducing steroid biosynthesis. Finally, real-time quantitative PCR and western blotting results showed that the expression trends of differentially expressed RNAs were consistent with the whole-transcriptome sequencing data.Discussion: this study provides more insights of comprehensive characterization of BTV-host interactome, and BTV infection and pathogenic mechanisms.

The ATP-binding cassette subfamily G member 2 (ABCG2) serves crucial roles in secreting riboflavin and biotin vitamins into the milk of cattle, mice, and humans, as well as in the transportation of xenotoxic and cytostatic drugs across the plasma membrane. However, the specific role of the ABCG2 gene in water buffaloes (Bubalus bubalis), especially its effect on milk fat synthesis in buffalo mammary epithelial cells (BuMECs), remains inadequately understood. In this study, the full-length CDS of the buffalo ABCG2 gene was isolated and identified from the mammary gland in buffaloes. A bioinformatics analysis showed a high degree of similarity in the transcriptional region, motifs, and conservative domains of the buffalo ABCG2 with those observed in other Bovidae species. The functional role of buffalo ABCG2 was associated with the transportation of solutes across lipid bilayers within cell membranes. Among the 11 buffalo tissues detected, the expression levels of ABCG2 were the highest in the liver and brain, followed by the mammary gland, adipose tissue, heart, and kidney. Notably, its expression in the mammary gland was significantly higher during peak lactation than during non-lactation. The ABCG2 gene was identified with five SNPs in river buffaloes, while it was monomorphic in swamp buffaloes. Functional experiments revealed that ABCG2 increased the triglyceride (TAG) content by affecting the expression of liposynthesis-related genes in BuMECs. The results of this study underscore the pivotal role of the ABCG2 gene in influencing the milk fat synthesis in BuMECs.

Porcine Sapelovirus (PSV) is widely prevalent in pig herds throughout the world and induces diarrhea, encephalomyelitis, respiratory tract symptoms, and reproductive disorders. However, the epidemiological and genetic evolution characteristics of PSV remain unclear in Yunnan Province. In this study, 1622 fecal samples were collected from pig farms in Yunnan Province. PSV and its co-infection rates with other pathogens were detected; then, the PSV VP1 gene was amplified and sequenced; and the genetic evolution characteristics of the VP1 gene were analyzed. The overall infection rate of PSV in Yunnan Province was 36.50%, and the differences among regions were significant (p < 0.05). The positive rates among different seasons were significantly different (p < 0.01), ranging from 73.33% (autumn) to 19.00% (summer). The PSV positive rate in diarrhea samples (47.26%) was significantly higher (p < 0.001) than that of non-diarrhea samples (31.77%). The co-infection rates of PSV with porcine rotavirus (PoRV) and PSV with porcine epidemic diarrhea virus (PEDV) were 5.07% and 3.04%. A total of 36 VP1 sequences were obtained, and the average identity among the 36 sequences was 85.3%, which was higher than that with other reference strains. Phylogenetic analysis revealed that all 36 PSV strains belonged to the PSV-1 genotype. The VP1 gene was under strong negative selection pressure (average dN/dS = 0.0838); however, the 95th amino acid was under positive selection pressure. Our study revealed the epidemiological, co-infection, and genetic evolution characteristics of PSV in pig herds of Yunnan Province, providing more data for preventing and controlling diarrhea pathogens in pigs.