Tannic acid alleviates ETEC K88-induced intestinal damage through regulating the p62-keap1-Nrf2 and TLR4-NF-κB-NLRP3 pathway in IPEC-J2 cells.

BACKGROUND: Tannic acid (TA), a naturally occurring polyphenol, has shown diverse potential in preventing intestinal damage in piglet diarrhea induced by Enterotoxigenic Escherichia coli (ETEC) K88. However, the protective effect of TA on ETEC k88 infection-induced post-weaning diarrhea and its potential mechanism has not been well elucidated. Therefore, an animal trial was carried out to investigate the effects of dietary supplementation with TA on the intestinal diarrhea of weaned piglets challenged with ETEC K88. In addition, porcine intestinal epithelial cells were used as an in vitro model to explore the mechanism through which TA alleviates intestinal oxidative damage and inflammation. RESULTS: The results indicated that TA supplementation (2 and 4 g kg-1) reduced diarrhea rate, enzyme activity (diamine oxidase [DAO] and Malondialdehyde [MAD]) and serum inflammatory cytokines concentration (TNF-α and IL-1ß) (P < 0.05) compared to the Infection group (IG), group in vivo. In vitro, TA treatment effectively alleviated ETEC-induced cytotoxicity, increased the expression of ZO-1, occludin and claudin-1 at both mRNA and protein levels. Moreover, TA pre-treatment increased the activity of antioxidant enzymes (such as T-SOD) and decreased serum cytokine levels (TNF-α and IL-1ß). Furthermore, TA increased cellular antioxidant capacity by activating the Nrf2 signaling pathway and decreased inflammatory response by down-regulating the expression of TLR4, MyD88, NF-kB and NLRP3. CONCLUSION: The present study showed that TA reduced the diarrhea rate of weaned piglets by restoring the intestinal mucosal mechanical barrier function, alleviating oxidative stress and inflammation. The underlying mechanism was achieved by modulating the p62-keap1-Nrf2 and TLR4-NF-κB-NLRP3 pathway. © 2024 Society of Chemical Industry.

Isolation and characterization of porcine epidemic diarrhea virus with a novel continuous mutation in the S10 domain.

Porcine epidemic diarrhea virus (PEDV), which re-emerged in China in 2010, has caused severe economic losses to the global pig industry. In this study, a PEDV strain, designated PEDV WMB, was isolated from piglets with severe diarrhea on a pig farm in Henan Province of China. Whole-genome sequencing and analysis revealed that the PEDV WMB strain belongs to subtype G2c and has a unique continuous mutation in the S10 antigenic epitope of the S protein. Moreover, the virus-neutralization (VN) test indicated that polyclonal antibodies against the S10 protein of other G1 and G2 strains showed reduced VN reactivity to PEDV WMB. The pathogenicity of PEDV WMB was further investigated in 3 day-old piglets. PEDV infection-related clinical symptoms and morphological lesions were observed and confirmed by histopathological and immunohistochemical examination (IHC). These results illustrated that continuous mutation of the S10 epitope might affect the immunogenicity or pathogenicity of PEDV, providing evidence of the need to monitor the genetic diversity of the virus and develop effective measures to prevent and control PEDV.

Prevalence and Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Diarrheic and Non-Diarrheic Calves from Ningxia, Northwestern China.

Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are significant parasitic gastrointestinal pathogens with global distribution in humans and domestic animals, including calves. The main symptoms of calf infection are severe diarrhea, dehydration, growth retardation, and sometimes even death. To date, there has been limited information on the prevalence of Cryptosporidium spp., G. duodenalis, and E. bieneusi infections in calves in Ningxia, China, especially between diarrheic and non-diarrheic calves. A total of 438 fecal samples were collected from diarrheic (201) and non-diarrheic (237) calves in Ningxia. PCR and DNA sequencing were used to find the prevalence of Cryptosporidium spp. at 46.8% (205/438), G. duodenalis at 16.9% (74/438), and E. bieneusi at 10.0% (44/438). The prevalence of Cryptosporidium spp. infection in diarrheic and non-diarrheic calves was 54.0% (128/237) and 38.3% (77/201), respectively, and statistical analysis showed a positive correlation between the prevalence of Cryptosporidium spp. infection and calf diarrhea (p < 0.01). However, in this study, there was no statistical correlation between the prevalence of G. duodenalis infection as well as E. bieneusi infection and calf diarrhea (p > 0.05). Furthermore, four known Cryptosporidium species were successfully identified by comparing them with SSU rRNA gene sequences, including C. parvum, C. bovis, C. ryanae, and C. andersoni. In addition, all 74 G. duodenalis-positive samples were identified as assemblage E by comparative analysis of bg gene sequences. Among the 44 E. bieneusi-positive samples sequenced in the present study, 4 distinct E. bieneusi genotypes were successfully identified by comparative analysis of ITS sequences, including 3 known genotypes (J, BEB4, and N) and 1 novel genotype, the latter of which was identified and designated as NX1. These findings indicated that the high genetic diversity and complex population structures of Cryptosporidium spp., G. duodenalis, and E. bieneusi in Ningxia diarrhea calves and non-diarrhea calves, which provide new data for understanding the epidemiological status of Cryptosporidium spp., G. duodenalis, and E. bieneusi in Ningxia calves.

Detection of the dominant pathogens in diarrheal calves of Ningxia, China in 2021-2022.

Introduction: Calf diarrhea is a complex disease that has long been an unsolved problem in the cattle industry. Ningxia is at the forefront of China in the scale of cattle breeding, and calf diarrhea gravely restricts the development of Ningxia's cattle industry. Methods: From July 2021 to May 2022, we collected diarrhea stool samples from calves aged 1-103 days from 23 farms in five cities in Ningxia, and performed PCR using specific primers for 15 major reported pathogens of calf diarrhea, including bacteria, viruses, and parasites. The effect of different seasons on the occurrence of diarrhea in calves was explored, the respective epidemic pathogens in different seasons were screened, and more detailed epidemiological investigations were carried out in Yinchuan and Wuzhong. In addition, we analyzed the relationship between different ages, river distributions and pathogen prevalence. Results: Eventually, 10 pathogens were detected, of which 9 pathogens were pathogenic and 1 pathogen was non-pathogenic. The pathogens with the highest detection rate were Cryptosporidium (50.46%), Bovine rotavirus (BRV) (23.18%), Escherichia coli (E. coli) K99 (20.00%), and Bovine coronavirus (BCoV) (11.82%). The remaining pathogens such as Coccidia (6.90%), Bovine Astrovirus (BoAstV) (5.46%), Bovine Torovirus (BToV) (4.09%), and Bovine Kobuvirus (BKoV) (3.18%) primarily existed in the form of mixed infection. Discussion: The analysis showed that different cities in Ningxia have different pathogens responsible for diarrhea, with Cryptosporidium and BRV being the most important pathogens responsible for diarrhea in calves in all cities. Control measures against those pathogens should be enforced to effectively prevent diarrhea in calves in China.

Rheological properties and volumetric isothermal expansivity of bamboo kraft black liquor with high solids content and low lignin content.

In this study, a certain percentage of lignin in original bamboo kraft black liquor (BKBL) was separated, and the residual BKBL with low lignin content was expected to be fed into the alkali recovery boiler to reduce the heat transfer load of the alkali recovery boiler. With the decrease in lignin content, the rheological properties/volumetric isothermal expansivity (VIE) of BKBL change. When the lignin content was 70% remaining in the original BKBL, the viscosity of BKBL with low lignin content is close to that of the passivated BKBL at the same solids content, the dynamic viscoelasticity is superior, and the VIE decreases by 57.2%. When the amount of desilication agent is 1.5%, the viscosity of BKBL with low lignin content did not change much, and the VIE increased sharply and was 62.7% higher than that of the passivated BKBL. Therefore, the combination of partial lignin separation process and sodium aluminate desilication process can effectively improve the ability of alkali recovery boiler to deal with BKBL and reduce the influence of "silicon interference".

25-hydroxycholesterol inhibits classical swine fever virus entry into porcine alveolar macrophages by depleting plasma membrane cholesterol.

Classical swine fever virus (CSFV) is an enveloped positive-sense RNA virus belonging to the Flaviviridae family. The virus utilizes cellular lipids and manipulates host lipid metabolism to ensure its replication, especially during virus invasion and replication steps. Therefore, identification of the molecular lipid metabolism pathways that are suitable targets is critical for the development of anti-CSFV therapeutics. In this study, we screened the anti-CSFV activity of 12 compounds targeting synthesis of cholesterol and fatty acids, cholesterol esters, and cholesterol transport. We found that 25-hydroxycholesterol (25HC), a regulator of cholesterol metabolism and transport, has potent anti-CSFV activity. Mechanistically, we showed that 25HC inhibited CSFV proliferation by blocking the entry of virions into porcine alveolar macrophages (3D4/21) by decreasing cholesterol abundance in the plasma membrane through activation of acyl-CoA:cholesterol acyltransferase (ACAT). Finally, we revealed that cholesterol 25-hydroxylase (CH25H), a redox enzyme that mediates 25HC production, also restricted CSFV infection via both enzyme activity-dependent and -independent mechanisms. Collectively, our results shed light on the mechanisms by which 25HC inhibits CSFV entry into cells and suggests a potential new therapeutic method against CSFV infection.

Antimicrobial activity of the recombinant peptide Melittin-Thanatin with three glycine to tryptophan mutations.

The antimicrobial peptide was considered an important target for developing novel antibacterial drugs. However, the unstable biological activity and the low antibacterial activity are challenges for the application of recombinant proteins. In this study, the fusion peptide of Melittin-Thanatin (MT) was designed and produced, and its derivative sequence (MT-W) was obtained by replacing three glycines (Gly, G) with tryptophan (Trp, W). The MT-W peptide were synthesized in Bacillus subtilis WB700 by EDDIE self-cleavage protein fusion. Compared with MT, MT-W exhibited 2-4 times higher antibacterial rate against Escherichia coli K88. In addition, MT-W showed lower cytotoxicity (IC50 > 300 mg·L-1) to the red blood cell, and more stable biological activities under the conditions of different temperatures (20, 30, 40, 50, 60, 70, 80, and 90 °C), pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) and different proteases. Especially, MT-W showed a broader antibacterial effect on three drug-resistant strains than florfenicol and oxytetracycline calcium. In conclusion, compared with MT, the MT-W showed increased antibacterial activity, stability, lower cytotoxicity, and broader antimicrobial effect. Therefore, it would become a promising alternative to conventional antibiotics.

An Investigation on the Influence of Operation Experience on Virtual Hazard Perception Using Wearable Eye Tracking Technology.

Poor electrical hazard recognition is a widespread issue in the production industry. Hazard perception has impacts workers' hazard recognition, causing them to experience unanticipated hazard exposure and suffer catastrophic injuries. To improve the factors of affecting hazard perception, the current study examined hazard recognition as an everyday visual search task. A comparative test was carried out combining the advantages and disadvantages of the two test methods. It was confirmed that the virtual image test data can replace the real image test data and demonstrate superior flexible settings performance, so the virtual image test method is used. A hazard perception test method based on wearable eye tracking technology was proposed to analyze the eye-tracking data (i.e., fixation, count, search duration, mean fixation duration, eye tracking, and hazard recognition performance feedback) were compared between experts in the field of electrical safety: skilled workers with at least five years of work experience and workers who had been on the job for less than a year. It was found that experts had a better hazard recognition accuracy and missed detection rate than other workers. Experts' hazards research track was more concised and paid less attention time. This advantage is most obvious in complex risk environments. The findings also suggest that workers who have different working years was not obvious visual search patterns other than the search duration. As can be seen the work experience is not an absolute factor in improving hazard perception. The present research will be useful to understand the influence of working years on hazard perception and provide a theoretical basis for corresponding training.

Rab22a cooperates with Rab5 and NS4B in classical swine fever virus entry process.

Classical swine fever virus (CSFV) is an ancient and economically important flavivirus that utilizes a Rab5-dependent endocytic pathway to enter host cells. Rab22a is a small GTPase that cooperates with Rab5 in the regulation of early endosome dynamics. Until now, the role of Rab22a in the flavivirus life cycle has been poorly defined. In this study, we systematically analyzed the role of Rab22a in CSFV proliferation and internalization using multiple viral replication analyses in combination with the overexpression, knockdown, and mutation of Rab22a, and found that Rab22a is involved in the entry process of CSFV. Confocal microscopy results showed that Rab22a colocalized with virus particles during the early phase of infection. Furthermore, by using glutathione S-transferase pull-down and co-immunoprecipitation assays, we verified the interaction between Rab22a and CSFV non-structural protein NS4B, and determined that NS4B can only bind to wild-type Rab22a, but not to the mutants Q64L and S19N. In addition, we explored the relationship between Rab22a, Rab5 and NS4B in CSFV internalization, and found out that these three proteins bind in early endosomes, and then through a Rab22a-Rab5-NS4B cascade allows the entry of CSFV. Taken together, our findings highlight the role of Rab proteins in CSFV internalization, and extend the understanding of the life cycle of flaviviruses.

ARF1 with Sec7 Domain-Dependent GBF1 Activates Coatomer Protein I To Support Classical Swine Fever Virus Entry.

Classical swine fever virus (CSFV), a positive-sense, enveloped RNA virus that belongs to the Flaviviridae family, hijacks cell host proteins for its own replication. We previously demonstrated that Golgi-specific brefeldin A (BFA) resistance factor 1 (GBF1), a regulator of intracellular transport, mediates CSFV infection. However, the molecular mechanism by which this protein regulates CSFV proliferation remains unelucidated. In this study, we constructed a series of plasmids expressing GBF1 truncation mutants to investigate their behavior during CSFV infection and found that GBF1 truncation mutants containing the Sec7 domain could rescue CSFV replication in BFA- and GCA (golgicide A)-treated swine umbilical vein endothelial cells (SUVECs), demonstrating that the effect of GBF1 on CSFV infection depended on the activity of guanine nucleotide exchange factor (GEF). Additionally, it was found that ADP ribosylation factors (ARFs), which are known to be activated by the Sec7 domain of GBF1, also regulated CSFV proliferation. Furthermore, we demonstrated that ARF1 is more important for CSFV infection than other ARF members with Sec7 domain dependence. Subsequent experiments established the function of coatomer protein I (COP I), a downstream effector of ARF1 which is also required for CSFV infection by mediating CSFV invasion. Mechanistically, inhibition of COP I function impaired CSFV invasion by inhibiting cholesterol transport to the plasma membrane and regulating virion transport from early to late endosomes. Collectively, our results suggest that ARF1, with domain-dependent GBF1 Sec7, activates COP I to facilitate CSFV entry into SUVECs. IMPORTANCE Classical swine fever (CSF), a highly contact-infectious disease caused by classical swine fever virus (CSFV) infecting domestic pigs or wild boars, has caused huge economic losses to the pig industry. Our previous studies have revealed that GBF1 and class I and II ARFs are required for CSFV proliferation. However, a direct functional link between GBF1, ARF1, and COP I and the mechanism of the GBF1-ARF1-COP I complex in CSFV infection are still poorly understood. Here, our data support a model in which COP I supports CSFV entry into SUVECs in two different ways, depending on the GBF1-ARF1 function. On the one hand, the GBF1-ARF1-COP I complex mediates cholesterol trafficking to the plasma membrane to support CSFV entry. On the other hand, the GBF1-ARF1-COP I complex mediates CSFV transport from early to late endosomes during the entry steps.

Tracing Clostridium perfringens strains from beef processing of slaughter house by pulsed-field gel electrophoresis, and the distribution and toxinotype of isolates in Shaanxi province, China.

The purpose of this study was to investigate the distribution and specify the transmission and cross-contamination of Clostridium perfringens (C. perfringens) in the beef slaughtering and butchering process. The prevalence of 21.2% (150/708) yielded 208 isolates of C. perfringens, including 80.8% type A and 19.2% type D, 0.4% (3/708) samples carried both type A and D strains, and 72.5% type D isolates carried both cpe and atyp.cpb2 genes. C. perfringens were identified through the whole slaughtering process but no type F (cpe and cpa isolates) was found. 69 isolates were further analyzed and classified into 28 PFGE genotypes and clade I contained 94.2% isolates and 24 PFGE genotypes, which showed the genetic diversity and epidemic correlation. Our study traced C. perfringens contamination along the handling processes and showed a gradually ascending contamination rate during the whole process, revealing widespread cross-contamination from the feces and hides of slaughtered cattle to the carcass in the slaughtering workshop, so as from tools and personnel to meat of the cutting workshops. Strains from different slaughterhouses (regions) have high homology, and type A is the predominant toxinotype. It is necessary to monitor and control several key points of cross-contamination during slaughtering process to reduce a risk of C. perfringens infection.

Extracellular vesicles originating from autophagy mediate an antibody-resistant spread of classical swine fever virus in cell culture.

Free spread is a classical mode for mammalian virus transmission. However, the efficiency of this transmission approach is generally low as there are structural barriers or immunological surveillances in the extracellular environment under physiological conditions. In this study, we systematically analyzed the spreading of classical swine fever virus (CSFV) using multiple viral replication analysis in combination with antibody neutralization, transwell assay, and electron microscopy, and identified an extracellular vesicle (EV)-mediated spreading of CSFV in cell cultures. In this approach, intact CSFV virions are enclosed within EVs and transferred into uninfected cells with the movement of EVs, leading to an antibody-resistant infection of the virus. Using fractionation assays, immunostaining, and electron microscopy, we characterized the CSFV-containing EVs and demonstrated that the EVs originated from macroautophagy/autophagy. Taken together, our results showed a new spreading mechanism for CSFV and demonstrated that the EVs in CSFV spreading are closely related to autophagy. These findings shed light on the immune evasion mechanisms of CSFV transmission, as well as new functions of cellular vesicles in virus lifecycles.Abbreviations: 3-MA: 3-methyladenine; CCK-8: Cell Counting Kit-8; CSF: classical swine fever; CQ: chloroquine; CSFV: classical swine fever virus; DAPI, 4-,6-diamidino-2-phenylindole; EVs: extracellular vesicles; hpi: h post infection; IEM: immunoelectron microscopy; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MOI: multiplicity of infection; MVs: microvesicles; ND50: half neutralizing dose; PCR: polymerase chain reaction; PBS: phosphate-buffered saline; SEC: size-exclusion chromatography; siRNA: small interfering RNA; TEM: transmission electron microscopy.

Chlorogenic acid inhibits apoptosis in thiram-induced tibial dyschondroplasia via intrinsic pathway.

Tibial dyschondroplasia (TD) is a common skeletal disease occurred in growth plate of fast-growing broilers. Thiram is a sort of chemical used for pesticide and fungicide. The excessive use of thiram increased the threat to animal and human health. In this study, we aimed to investigate the therapeutic mechanism of chlorogenic acid (CGA) on thiram-induced tibial dyschondroplasia. Broiler chickens were divided into three different groups, e.g., control, TD, and CGA. CGA was administrated after the induction of TD from 4th day to 7th day. Biochemical analysis was performed to detect the content of calcium (Ca) and phosphorus (P). Histological changes and degradation of extracellular matrix were observed through hematoxylin-eosin (H & E) and Masson staining. To further determine the mechanism, TUNEL staining and western blot were also performed to detect the apoptosis changes in growth plate of all groups. The results showed the disproportionation of Ca and P content and upregulation of apoptosis during the development of TD. But, after the administration of CGA, the ratio of Ca:P was upregulated, and the apoptosis was also downregulated. The current study shows the toxic effect of thiram on chickens and suggests that CGA is associated with a mechanism that plays a significant role in apoptosis induced by thiram in poultry industry.

Risk of Environmental Exposure to H7N9 Influenza Virus via Airborne and Surface Routes in a Live Poultry Market in Hebei, China.

Environmental transmission of viruses to humans has become an early warning for potential epidemic outbreaks, such as SARS-CoV-2 and influenza virus outbreaks. Recently, an H7N9 virus, A/environment/Hebei/621/2019 (H7N9), was isolated by environmental swabs from a live poultry market in Hebei, China. We found that this isolate could be transmitted by direct contact and aerosol in mammals. More importantly, after 5 passages in mice, the virus acquired two adaptive mutations, PB1-H115Q and B2-E627K, exhibiting increased virulence and aerosol transmissibility. These results suggest that this H7N9 virus might potentially be transmitted between humans through environmental or airborne routes.

ARFGAP1 binds to classical swine fever virus NS5A protein and enhances CSFV replication in PK-15 cells.

Classical swine fever virus (CSFV), an enveloped virus belonging to the genus Pestivirus of the Flaviviridae family, utilizes cell host factors for its own replication. ARFGAP1, GTPase activating protein of ADP-ribosylation factor 1, regulates COP I vesicle formation and function in cells and is involved in the life cycle of several viruses. However, the effect of ARFGAP1 on the infection of CSFV has not been illustrated. Here we showed that inhibition of ARFGAP1 either by QS11 or by lentivirus-mediated silencing repressed CSFV replication. While, subsequent experiments revealed that CSFV production were increased in cells with sufficient ARFGAP1 expression. However, ARFGAP1 was not involved in CSFV binding, entry, access to cell vesicles, and RNA replication during the early stages of infection. Then, we showed that ARFGAP1 interacted with the viral protein of NS5A, measured by immunoprecipitation, GST-pulldown, and confocal microscopy assays. Furthermore, we revealed that ARFGAP1 could alleviated CSFV NS5A-induced endoplasmic reticulum stress (ERS). Altogether, these results demonstrate that ARFGAP1, a NS5A binding protein, is involved in CSFV replication.

Genetic Diversity and Prevalence of Porcine Circovirus Type 2 in China During 2000-2019.

As the major pathogen for porcine circovirus-associated disease (PCVAD), porcine circovirus type 2 (PCV2) is no longer treated as an emerging virus anymore. The wide distribution of PCV2 infection in China causes huge economic losses in the swine industry. Currently, it is generally believed that PCV2 has eight genotypes (PCV2a to PCV2h), with PCV2a, PCV2b, and PCV2d being widely distributed. To comprehensively explore the genetic diversity and prevalence of PCV2 in China, PCV-2 sequences submitted from China in the GenBank database were retrieved. With a total of 714 PCV2 strains were retrieved, we found that early-submitted PCV2 sequences were mainly collected from coastal provinces in the southeast part of China, which may indicate PCV2 was initially circulating in those regions. From 2002 to 2008, PCV2b was the dominant prevalent genotype in those retrieved sequences. From 2009, PCV2d became the dominant genotype in those sequences, dropping a hint that a potential shift of PCV2b to PCV2d might occur in 2009, which is similar to the patterns at the global level. In addition to the PCV2a, PCV2b, and PCV2d genotypes, novel strains were also characterized. We further revealed that the amino acid sequences consistency of PCV2a Cap is higher than those in other genotypes. Together, this study provided clues for the possible prevalent genotypes and dynamics of genetic diversity in China from 2000 to 2019.

Diverse Gene Cassette Arrays Prevail in Commensal Escherichia coli From Intensive Farming Swine in Four Provinces of China.

Multiple-drug resistance bacteria containing antimicrobial resistance genes (ARGs) are a concern for public health. Integrons are bacterial genetic elements that can capture, rearrange, and express mobile gene cassettes responsible for the spread of ARGs. Few studies link genotype and phenotype of swine-related ARGs in the context of mobile gene cassette arrays among commensal Escherichia coli (E. coli) in nonclinical livestock isolates from intensive farms. In the present study, a total of 264 isolates were obtained from 330 rectal swabs to determine the prevalence and characteristics of antibiotic-resistant gene being carried by commensal E. coli in the healthy swine from four intensive farms at Anhui, Hebei, Shanxi, and Shaanxi, in China. Antimicrobial resistance phenotypes of the recovered isolates were determined for 19 antimicrobials. The E. coli isolates were commonly nonsusceptible to doxycycline (75.8%), tetracycline (73.5%), sulfamethoxazole-trimethoprim (71.6%), amoxicillin (68.2%), sulfasalazine (67.1%), ampicillin (58.0%), florfenicol (56.1%), and streptomycin (53.0%), but all isolates were susceptible to imipenem (100%). Isolates [184 (69.7%)] exhibited multiple drug resistance with 11 patterns. Moreover, 197 isolates (74.6%) were detected carrying the integron-integrase gene (intI1) of class 1 integrons. A higher incidence of antimicrobial resistance was observed in the intI1-positive E. coli isolates than in the intI1-negative E. coli isolates. Furthermore, there were 17 kinds of gene cassette arrays in the 70 integrons as detected by sequencing amplicons of variable regions, with 66 isolates (94.3%) expressing their gene cassettes encoding for multiple drug resistance phenotypes for streptomycin, neomycin, gentamicin, kanamycin, amikacin, sulfamethoxazole-trimethoprim, sulfasalazine, and florfenicol. Notably, due to harboring multiple, hybrid, and recombination cassettes, complex cassette arrays were attributed to multiple drug resistance patterns than simple arrays. In conclusion, we demonstrated that the prevalence of multiple drug resistance and the incidence of class 1 integrons were 69.7 and 74.6% in commensal E. coli isolated from healthy swine, which were lower in frequency than that previously reported in China.

A novel PCV2 ORF5-interacting host factor YWHAB inhibits virus replication and alleviates PCV2-induced cellular response.

Porcine circovirus type 2 (PCV2) infection causes porcine circovirus associated diseases (PCVAD) worldwide. Identification of host factors that interact with viral proteins is a fundamental step to understand the pathogenesis of PCV2. Our previous study reported that ORF5, a newly identified PCV2 viral protein supports PCV2 replication and interacts with multiple host factors. Here, we showed that a host factor YWHAB is an ORF5-interacting protein and plays essential roles during PCV2 infection. By using protein-protein interaction assays, we confirmed that YWHAB directly interacts with PCV2-ORF5 protein. We further showed that YWHAB expression was potently induced upon ORF5 overexpression and PCV2 infection. Remarkably, we found that the YWHAB strongly inhibited PCV2 replication, suggesting its role in defending PCV2 infection. By using the ectopic overexpression and gene knockdown approaches, we revealed that YWHAB inhibits PCV2-induced endoplasmic reticulum stress (ERS), autophagy, reactive oxygen species (ROS) production and apoptosis, suggesting its vital role in alleviating PCV2-induced cellular damage. Together, this study demonstrated that an ORF5-interacting host factor YWHAB affects PCV2 infection and PCV2-induced cellular response, which expands the current understanding of YWHAB biological function and might serves as a new therapeutic target to manage PCV2 infection-associated diseases.

Rab1b-GBF1-ARFs mediated intracellular trafficking is required for classical swine fever virus replication in swine umbilical vein endothelial cells.

Classical swine fever virus (CSFV), a plus-sense RNA virus, utilizes host intracellular membrane organelles for its replication. Our previous studies have shown that disruption of the intracellular membrane-trafficking events can inhibit CSFV replication. However, the underlying mechanism of this process in CSFV infection has not been elucidated. To determine the role of Golgi-associated anterograde and retrograde trafficking in CSFV replication, we revealed the effect of vesicular transport between Golgi and ER inhibitors Brefeldin A (BFA) and 2,2-methyl-N-(2,4,6,-trimethoxyphenyl) dodecanamide (CI-976), the GBF1 inhibitor golgicide A (GCA) on virus production. Our results showed that disruption of vesicular trafficking by BFA, CI-976, and GCA significantly inhibited CSFV infection. Subsequent experiments revealed that knockdown of Rab1b by lentiviruses and negative-mutant Rab1b-N121I transfection inhibited CSFV infection. Furthermore, we showed that the Rab1b downstream vesicular component effectors GBF1, and class I and class II ADP-ribosylation factors (ARFs) were also involved in virus replication. In addition, confocal microscopy assay showed that CSFV infection disrupted the Golgi apparatus resulting in extended Golgi distribution around the nucleus. We also showed that cell secretory pathway, measured using Gaussia luciferase flash assay, was blocked in CSFV infected cells. Taken together, these findings demonstrate that CSFV utilizes Rab1b-GBF1-ARFs mediated trafficking to promote its own replication. These findings also provide new insights into the intracellular trafficking pathways utilized for CSFV life cycle.

Rab18 binds to classical swine fever virus NS5A and mediates viral replication and assembly in swine umbilical vein endothelial cells.

Classical swine fever virus (CSFV), a positive-sense RNA virus, hijacks cell host proteins for its own replication. Rab18, a small Rab GTPase, regulates intracellular membrane-trafficking events between various compartments in cells and is involved in the life cycle of multiple viruses. However, the effect of Rab18 on the production of CSFV remains uncertain. In this study, we showed that knockdown of Rab18 by lentiviruses inhibited CSFV production, while overexpression of Rab18 by lentiviruses enhanced CSFV production. Subsequent experiments revealed that the negative-mutant Rab18-S22 N inhibited CSFV infection, while the positive-mutant Rab18-Q67 L enhanced CSFV infection. Furthermore, we showed that CSFV RNA replication and virion assembly, measured by real-time fluorescence quantitative PCR (RT-qPCR), indirect immunofluorescence assay (IFA), and confocal microscopy, were reduced in cells lacking Rab18 expression. In addition, co-immunoprecipitation, GST-pulldown, and confocal microscopy assays revealed that Rab18 bound to the viral protein NS5A. Further, NS5A was shown to be redistributed in Rab18 knockdown cells. Taken together, these findings demonstrate Rab18 as a novel host factor required for CSFV RNA replication and particle assembly by interaction with the viral protein NS5A.