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A multiplex polymerase chain reaction (mPCR) assay was developed to detect and distinguish feline panleukopenia virus (FPV), feline bocavirus (FBoV) and feline astrovirus (FeAstV). Three pairs of primers were designed based on conserved regions in the genomic sequences of the three viruses and were used to specifically amplify targeted fragments of 237 bp from the VP2 gene of FPV, 465 bp from the NP1 gene of FBoV and 645 bp from the RdRp gene of FeAstV. The results showed that this mPCR assay was effective, because it could detect at least 2.25-4.04 × 104 copies of genomic DNA of the three viruses per μl, was highly specific, and had a good broad-spectrum ability to detect different genotypes of the targeted viruses. A total of 197 faecal samples that had been screened previously for FeAstV and FBoV were collected from domestic cats in northeast China and were tested for the three viruses using the newly developed mPCR assay. The total positive rate for these three viruses was 59.89% (118/197). From these samples, DNA from FPV, FBoV and FeAstV was detected in 73, 51 and 46 faecal samples, respectively. The mPCR testing results agreed with the routine PCR results with a coincidence rate of 100%. The results of this study show that this mPCR assay can simultaneously detect and differentiate FPV, FBoV and FeAstV and can be used as an easy, specific and efficient detection tool for clinical diagnosis and epidemiological investigation of these three viruses.

Porcine epidemic diarrhea virus (PEDV) is a significant pathogen affecting swine, causing severe economic losses worldwide. This study explores the regulatory role of miRNA-328-3p to ZO-1 expression and its impact on PEDV proliferation via the PLC-β1-PKC pathway in IPEC-J2 cells. We found that miRNA-328-3p can target ZO-1, influencing its expression and subsequently affecting the integrity of tight junctions in the cells. Overexpression of PLC-β1, combined with miRNA-328-3p silencing, enhanced ZO-1 expression, while PLC-β1 knockdown combined with miRNA-328-3p overexpression inhibited ZO-1 expression. Furthermore, PLC-β1 overexpression increased both viral genome expression and PEDV titers, whereas its silencing had the opposite effect. Notably, our data indicated a negative correlation between PLC-β1 and PKC expression, and PKC silencing attenuated the upregulatory effect of PLC-β1 on ZO-1. These findings suggest that PLC-β1 modulates ZO-1 expression through the PKC pathway, providing new insights into the molecular mechanisms of PEDV infection and potential therapeutic targets.

Feline astrovirus (FeAstV) which belonged to the genus Mamastrovirus was first identified in the feces of kittens with diarrhea in the USA in 1981 by electron microscopy, and had been reported in many countries. Presently, there are no any reports of the circulation of FeAstV in mainland China. We performed this study to investigate the apparent prevalence and genetic variability of FeAstV infected in cats in mainland China for the first time. We tested fecal samples of 105 cats with diarrhea and 92 asymptomatic cats in five cities in northeast China by RT-PCR targeting RNA-dependent RNA polymerase (RdRp) gene of FeAstV, and analyzed sequences variability and phylogenetic evolution based on the complete capsid gene of FeAstV strains obtained from positive samples. The overall prevalence of FeAstV was 23.4% (46/197) of which 38 were tested in cats with diarrhea (36.2%, 38/105) and 8 were in asymptomatic cats (8.7%, 8/92). Mixed infection with other enteroviruses including feline parvovirus (FPV), feline bocavirus (FBoV) and feline kobuvirus (FeKoV) was found in 38 FeAstV-positive samples. Phylogenetic analysis based on the complete capsid gene revealed all FeAstV strains were divided into two different groups with a 0.454±0.016 of mean amino acid genetic distance between two groups, suggesting that FeAstVs should be classified into two different genotype species. This study provided the first molecular evidence that FeAstV with considerable genetic diversity was circulating in northeast China, and analyzed genetic variability and classification of FeAstVs for the first time.

As essential transfer carriers for cell-to-cell communication and genetic material, exosomes carry microRNAs that participate in the regulation of various biological processes. MicroRNAs are a type of single-stranded noncoding RNA that bind to specific target gene mRNAs to degrade or inhibit their translation, thereby regulating target gene expression. Although it is known that a variety of microRNAs are involved in the viral infection process, there are few reports on specific microRNAs involved in porcine epidemic diarrhea virus (PEDV) infection. In this study, we isolated and identified exosomes in PEDV-infected Vero E6 cells. Using transcriptomics technology, we found that miRNA-328-3p was significantly downregulated in exosomes following PEDV infection. Moreover, exosomal miRNA-328-3p inhibited infection by PEDV by targeting and inhibiting tight junction protein 3 (TJP-3/ZO-3) in recipient cells. Our findings provide evidence that, after infecting cells, PEDV downregulates expression of miRNA-328-3p, and the resulting reduced inhibition of the target protein ZO-3 helps to enhance PEDV infection. These results provide new insight for understanding the regulatory mechanism of PEDV infection.

This study aimed to investigate the effects of yeast cultures on the antioxidant capacity, rumen fermentation, and growth performance of goats in the summer. An in vitro experiment was conducted using yeast culture supplemented at 0% (control), 0.6% (test 1), 0.9% (test 2), and 1.2% (test 3) of the dry matter (DM) weight of the basal diet. With a 24 h fermentation, the pH value; the total short-chain fatty acid, acetic acid, propionic acid, and butyric acid concentrations; and the degradability of the DM, the neutral detergent fiber, and the acid detergent fiber were significantly increased (p < 0.05) in tests 2 and 3 compared with the control group. In the feeding experiment, thirty-six crossbreed goats aged 3.0 ± 0.5 months with a body weight of 11.08 ± 1.41 kg were divided, and the yeast culture was supplemented at 0% (control), 0.90% (test 1), and 1.20% (test 2) of the basal diet. Similar effects on rumen fermentation parameters were obtained in test 1 and 2 groups compared to the in vitro experiment. Moreover, the dry matter intake, average daily gain, serum total antioxidant capacity, and the activities of total superoxide dismutase and glutathione peroxidase were significantly higher and the malondialdehyde concentration was significantly lower (p < 0.05) in tests 1 and 2 compared with the control. The results indicated that yeast culture (0.90%) could improve the antioxidant capacity, rumen fermentation, and growth performance of goats in summer. The optimal supplementation concentration is 0.90% DM.

Background Bovine ephemeral fever virus (BEFV), the causative agent of bovine ephemeral fever, is an economically important pathogen of cattle and water buffalo. MicroRNAs (miRNAs) are endogenous 21-23 nt small non-coding RNA molecules that binding to a multiple of target mRNAs and functioning in the regulation of viral replication including the miRNA-mediated antiviral defense. However, the reciprocal interaction between bovine ephemeral fever virus replication and host miRNAs still remain poorly understood. The aim of our study herein was to investigate the exact function of miR-3470b and its molecular mechanisms during BEFV infection. Results In this study, we found a set of microRNAs induced by BEFV infection using small RNA deep sequencing, and further identified BEFV infection could significantly up-regulate the miR-3470b expression in Baby Hamster Syrian Kidney cells (BHK-21) after 24 h and 48 h post-infection (pi) compared to normal BHK-21 cells without BEFV infection. Additionally, the target association between miR-3470b and mitochondrial antiviral signaling protein (MAVS) was predicted by target gene prediction tools and further validated using a dual-luciferase reporter assay, and the expression of MAVS mRNA and protein levels was negatively associated with miR-3470b levels. Furthermore, the miR-3470b mimic transfection significantly contributed to increase the BEFV N mRNA, G protein level and viral titer, respectively, whereas the miR-3470b inhibitor had the opposite effect on BEFV replication. Moreover, the overexpression of MAVS or silencing of miR-3470b by its inhibitors suppressed BEFV replication, and knockdown of MAVS by small interfering RNA also promoted the replication of BEFV. Conclusions Our findings is the first to reveal that miR-3470b as a novel host factor regulates BEFV replication via directly targeting the MAVS gene in BHK-21 cells and may provide a potential strategy for developing effective antiviral therapy.

The aim of this study was to investigate the protective effects of cepharanthine (CEP) on inflammation in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in vitro and a LPS-induced lung injury model in vivo . RAW264.7 cells were treated with various concentrations of CEP for 1 h followed by incubation with or without 1 μg/ml LPS for 18 h. TNF-α, IL-6, and IL-1β in the supernatants were measured by ELISA. Nuclear factor-κB (NF-κB) and mitogen-activated protein kinase pathways were analyzed by Western blot. Mice were randomly divided into control group, LPS group, CEP + LPS group, and dexamethasone + LPS group. A male BALB/c mouse model of acute lung injury was induced by LPS. Bronchoalveolar lavage fluid was collected for inflammatory cell count and cytokine assays. Histopathologic examination was performed on mice that were not subjected to bronchoalveolar lavage fluid collection. CEP dose-dependently inhibited the release of TNF-α, IL-6, and IL-1β in LPS-stimulated RAW264.7 cells. Significantly, CEP dose-dependently suppressed NF-κB activation, IκBα degradation, and phosphorylation of ERK, JNK, and p38 induced by LPS. In vivo , it was also observed that CEP attenuated lung histopathologic changes and down-regulated the level of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, in the mouse acute lung injury model. These results suggest that CEP potentially decreases inflammation in vitro and in vivo and might be a therapeutic agent against inflammatory diseases.

Cats infected with feline calicivirus (FCV) often display oral ulcers and inflammation of the upper respiratory tract, which can lead to death in severe cases. Antiviral therapy is one of the most effective ways to control FCV infection. Natural compounds in Chinese herbal medicines and medicinal plants provide abundant resources for research on antiviral drugs. In this study, we found that icariin (ICA), formononetin (FMN) and caffeic acid phenethyl ester (CPAE) show low cytotoxicity towards F81 cells, that the three natural compounds have apparent antiviral effects on FCV in vitro, and that they can inhibit different FCV strains. Then, we found that ICA and FMN mainly function in the early stage of FCV infection, while CAPE can function in both the early and late stages of FCV infection. Finally, we found that ICA has an antagonistic effect on FMN and CAPE in FCV infection, and FMN has a synergistic effect with CAPE against FCV infection. Our results showed that ICA, FMN and CAPE may be potential drug candidates for FCV-induced diseases.

Background Bocaviruses have been reported to cause respiratory tract infection and gastroenteritis in most animal species. In cats, different genotype bocaviruses have been identified in USA, Japan, Hong Kong and Portugal. However, the clear relationship between the clinical symptoms and FBoV infection is unknown, and the prevalence of FBoV and the distribution of FBoV genotypes in China are still unclear. Results In this study, 197 fecal samples from cats with diarrhea ( n  = 105) and normal cats ( n  = 92) were collected in different regions between January 2016 and November 2017 and investigated using PCR targeting different FBoV genotypes. Screening results showed that 51 of 197 samples (25.9%) were positive for FBoV, and a higher positive rate was observed in cats with diarrhea (33.3%, 35/105) than in normal cats (17.4%, 16/92). Of these FBoV-positive samples, 35 were identified as FBoV-1, 12 as FBoV-2 and 4 as coinfection of FBoV-1 and FBoV-2. A phylogenetic analysis based on partial NS1 gene indicated that 24 sequences from randomly selected FBoV-positive samples were divided into 2 different FBoV groups: FBoV-1 and FBoV-2. Furthermore, 6 strains were randomly selected, and the complete genome was sequenced and analyzed. These strains exhibited the typical genome organization of bocavirus and were closely related to FBoV. Two FBoV-2 identified strains shared high homologies with FBoV-2 reference strains based on the complete genome and entire encoding gene, but lower identities were exhibited in the NP1 and VP1 regions for the other 4 FBoV-1 identified strains compared with FBoV-1 reference strains. Conclusion These findings demonstrate that genetically diverse FBoV-1 and FBoV-2 widely circulate in cats in Northeast China and that FBoV-1 is more prevalent. The high prevalence of FBoV in cats with diarrhea symptoms suggests that FBoV infection may be associated with diarrhea in cats.

A cross-priming isothermal amplification (CPA) assay was developed for detection of feline herpesvirus type 1 (FHV-1). In this assay, the target fragment of the FHV-1 glycoprotein B gene is amplified rapidly by Bst DNA polymerase at a constant temperature (63 °C, 45 min), using a simple thermostat. The assay had no cross-reactions with four types of feline viruses, and the detection limit was 100 copies/μl. The positive rate of clinical samples from CPA was 100% consistent with qPCR but higher than ordinary PCR, indicating its superiority to ordinary PCR. Visualization was achieved using SYBR Green I dye.