Licorice extract inhibits porcine epidemic diarrhea virus in vitro and in vivo.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and even death in piglets, resulting in significant economic losses to the pig industry. Because of the ongoing mutation of PEDV, there might be variations between the vaccine strain and the prevailing strain, causing the vaccine to not offer full protection against different PEDV variant strains. Therefore, it is necessary to develop anti-PEDV drugs to compensate for vaccines. This study confirmed the anti-PEDV effect of licorice extract (Le) in vitro and in vivo. Le inhibited PEDV replication in a dose-dependent manner in vitro. By exploring the effect of Le on the life cycle of PEDV, we found that Le inhibited the attachment, internalization, and replication stages of the virus. In vivo, all five piglets in the PEDV-infected group died within 72 h. In comparison, the Le-treated group had a survival rate of 80 % at the same time, with significant relief of clinical symptoms, pathological damage, and viral loads in the jejunum and ileum. Our results suggested that Le can exert anti-PEDV effects in vitro and in vivo. Le is effective and inexpensive; therefore it has the potential to be developed as a new anti-PEDV drug.

The outer membrane protein of Fusobacterium necrophorum, 43K OMP, stimulates inflammatory cytokine production through nuclear factor kappa B activation.

OBJECTIVE: Fusobacterium necrophorum causes bovine hepatic abscess, foot rot, mastitis, and endometritis. The 43 kDa outer membrane protein (43 K OMP) of F. necrophorum is a porin protein that plays an important role in infections by this bacterium, but the biological function and the pathogenesis of this protein are largely unknown. METHODS: In this study, we investigated the role of the 43 K OMP in bacterial infection of bovine mammary epithelial cells (MAC-T cells) by Tandem Mass Tag proteomic analysis. The RAW264.7 cells were incubated with recombinant 43 K OMP (12.5 µg/mL) for 2 h, 4 h, 6 h, and 12 h, and then the inflammatory related protein and inflammatory cytokine production were measured by Western blot analysis and ELISA, the mRNA expression levels of inflammatory cytokine were measured by Real-Time PCR. RESULTS: Proteomic analysis results demonstrated there were 224 differentially expressed proteins in the MAC-T cells stimulated with the 43 K OMP compared with control, and 118 proteins were upregulated and 106 proteins were downregulated. These differentially expressed proteins were mainly involved in NF-kappa B signaling, bacterial invasion of epithelial cells, cell adhesion, complement and coagulation cascades. The top six differentially expressed proteins were; MMP9, PLAU, STOM, PSMD13, PLAUR, and ITGAV, which were involved in a protein-protein interaction network. Furthermore, TLR/MyD88/NF-κB pathway related proteins and inflammatory cytokines (IL-6, TNF-α, and IL-1ß) were assessed by Western blot analysis and ELISA. Results showed the 43 K OMP to enhance the expression of TLR4 protein at 2 h (P < 0.01) and the MyD88 protein at 4 h (P < 0.05) post-stimulation, and to decrease IκBα expression at 4 h, 6 h and 12 h (P < 0.05) post-infection, as well as induce phosphorylation at Ser536 (P < 0.01). Levels of IL-6, IL-1ß, and TNF-α in the supernatants of mouse macrophages were increased (P < 0.05), as were mRNA expression levels of IL-6, IL-1ß, and TNF-α (P < 0.05), while IL-4 mRNA expression was decreased (P < 0.05). CONCLUSIONS: Taken together, these results suggested the important role for 43 K OMP in F. necrophorum infection, promoting the production of pro-inflammatory cytokines (IL-6 and TNF-α) by activation of the TLR/MyD88/NF-κB pathway. These findings provided a theoretical basis for a better understanding of the pathogenesis of F. necrophorum infection.

Coronavirus Porcine Epidemic Diarrhea Virus Nucleocapsid Protein Interacts with p53 To Induce Cell Cycle Arrest in S-Phase and Promotes Viral Replication.

Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication (P < 0.05). S-phase arrest was dependent on the N protein nuclear localization signal S71NWHFYYLGTGPHADLRYRT90 and the interaction between N protein and p53. In the nucleus, the binding of N protein to p53 maintained consistently high-level expression of p53, which activated the p53-DREAM pathway. The key domain of the N protein interacting with p53 was revealed to be S171RGNSQNRGNNQGRGASQNRGGNN194 (NS171-N194), in which G183RG185 are core residues. NS171-N194 and G183RG185 were essential for N-induced S-phase arrest. Moreover, small molecular drugs targeting the NS171-N194 domain of the PEDV N protein were screened through molecular docking. Hyperoside could antagonize N protein-induced S-phase arrest by interfering with interaction between N protein and p53 and inhibit viral replication (P < 0.05). The above-described experiments were also validated in porcine intestinal cells, and data were in line with results in Vero E6 cells. Therefore, these results reveal the PEDV N protein interacts with p53 to activate the p53-DREAM pathway, and subsequently induces S-phase arrest to create a favorable environment for virus replication. These findings provide new insight into the PEDV-host interaction and the design of novel antiviral strategies against PEDV. IMPORTANCE Many viruses subvert the host cell cycle to create a cellular environment that promotes viral growth. PEDV, an emerging and reemerging coronavirus, has led to substantial economic loss in the global swine industry. Our study is the first to demonstrate that PEDV N-induced cell cycle arrest during the S-phase promotes viral replication. We identified a novel mechanism of PEDV N-induced S-phase arrest, where the binding of PEDV N protein to p53 maintains consistently high levels of p53 expression in the nucleus to mediate S-phase arrest by activating the p53-DREAM pathway. Furthermore, a small molecular compound, hyperoside, targeted the PEDV N protein, interfering with the interaction between the N protein and p53 and, importantly, inhibited PEDV replication by antagonizing cell cycle arrest. This study reveals a new mechanism of PEDV-host interaction and also provides a novel antiviral strategy for PEDV. These data provide a foundation for further research into coronavirus-host interactions.

Potential of bacteriophages as disinfectants to control of Staphylococcus aureus biofilms.

BACKGROUND: Staphylococcus aureus is the causative agent of chronic mastitis, and can form a biofilm that is difficult to completely remove once formed. Disinfectants are effective against S. aureus, but their activity is easily affected by environmental factors and they are corrosive to equipment and chemically toxic to livestock and humans. Therefore, we investigated the potential utility of a bacteriophage as a narrow-spectrum disinfectant against biofilms formed by S. aureus. In this study, we isolated and characterized bacteriophage vB_SauM_SDQ (abbreviated to SDQ) to determine its efficacy in removing S. aureus biofilms. RESULTS: SDQ belongs to the family Myoviridae and consists of a hexagonal head, long neck, and short tail. This phage can sterilize a 109 CFU/mL culture of S. aureus in 12 h and multiply itself 1000-fold in that time. Biofilms formed on polystyrene, milk, and mammary-gland tissue were significantly reduced after SDQ treatment. Fluorescence microscopy and scanning electron microscopy showed that SDQ destroyed the biofilm structure. Moreover, the titer of SDQ remained relatively high after the lysis of the bacteria and the removal of the biofilm, exerting a continuous bacteriostatic effect. SDQ also retained its full activity under conditions that mimic common environments, i.e., in the presence of nonionic detergents, tap water, or organic materials. A nonionic detergent (Triton X-100) enhanced the removal of biofilm by SDQ. CONCLUSIONS: Our results suggest that SDQ, a specific lytic S. aureus phage, can be used to control biofilm infections. SDQ maintains its full activity in the presence of nonionic detergents, tap water, metal chelators, and organic materials, and can be used in combination with detergents. We propose this phage as a narrow-spectrum disinfectant against S. aureus, to augment or supplement the use of broad-spectrum disinfectants in the prevention and control of the mastitis and dairy industry contamination caused by S. aureus.

Isolation and characterization of a variant subgroup GII-a porcine epidemic diarrhea virus strain in China.

BACKGROUND: Highly virulent variants of porcine epidemic diarrhea virus (PEDV) have been closely associated with recent outbreaks of porcine epidemic diarrhea (PED) in China, which have resulted in severe economic losses to the pork industry. METHODS: In the current study, the variant PEDV strain HM2017 was isolated and purified and a viral growth curve was constructed according to the median tissue culture infective dose (TCID50). HM2017 were amplify with RT-PCR and analyzed by phylogeny analysis. Animal pathogenicity experiment was carried to evaluate the HM2017 clinical assessment. RESULTS: Genome-based phylogenetic analysis revealed that PEDV strain HM2017 was clustered into the variant subgroup GII-a that is currently circulating in pig populations in China. The highest median tissue culture infectious dose of strain HM2017 after 15 passages in Vero cells was 1.33 × 107 viral particles/mL. Strain HM2017 was highly virulent to suckling piglets, which exhibited clinical symptoms at 12 h post-infection (hpi) (i.e., weight loss at 12-84 hpi, increased body temperatures at 24-48 hpi, high viral loads in the jejunum and ileum, and 100% mortality by 84 hpi). CONCLUSION: The present study reports a variant subgroup GII-a PEDV HM2017 strain in China and characterize its pathogenicity. PEDV strain HM2017 of subgroup GII-a presents a promising vaccine candidate for the control of PED outbreaks in China.

Screening of BHK-21 cellular proteins that interact with outer membrane protein 43K OMP of Fusobacterium necrophorum.

Fusobacterium necrophorum is a Gram negative, spore-free, anaerobic bacterium that can cause pyogenic and necrotic infections in animals and humans. It is a major bovine pathogen and causes hepatic abscesses, foot rot, and necrotic laryngitis. The 43K OMP of F. necrophorum is an outer membrane protein with molecular weight of 43 kDa, exhibiting similarity to pore-forming proteins of other Fusobacterium species that plays an important role in bacterial infections. However, the role of 43K OMP in F. necrophorum adhesion remains unknown. In this study, we evaluated whether the 43K OMP of F. necrophorum mediates adhesion to BHK-21 cells and performed a preliminary screen of the proteins that interact with 43K OMP of F. necrophorum by immunoprecipitation-mass spectrometry. The results showed that the natural 43K OMP and recombinant 43K OMP could bind to BHK-21 cells, and preincubation of F. necrophorum with an antibody against the recombinant 43K OMP of F. necrophorum decreased binding to BHK-21 cells. Seventy differential interacting proteins were successfully screened by immunoprecipitation-mass spectrometry. Among these seventy differential interacting proteins, seven cell membrane proteins and four extracellular matrix proteins shown to be relevant to bacteria adhesion through subcellular localization and single-molecule function analysis. These data increase our understanding of the pathogenesis of F. necrophorum and provide a new theoretical basis for the design of antimicrobial drugs against F. necrophorum.

Epidemiology of porcine epidemic diarrhea virus among Chinese pig populations: A meta-analysis.

BACKGROUND: Porcine epidemic diarrhea results from infection with porcine epidemic diarrhea virus (PEDV). It is an acute and highly contagious enteric disease in swine characterized by watery diarrhea and vomiting. Here, we performed a systematic review and meta-analysis in order to assess the prevalence of PEDV infection in pig populations from mainland China. METHODS: We conducted a literature search on the prevalence of PEDV infection in pigs between Jan 1, 1988 and Aug 20, 2018 in English and Chinese databases, including PubMed, Google scholar, Cochrane library, Clinical Trials, VIP, CNKI and WanFang database. Selections were made based on the title and the abstract of paper, and duplicated literature was excluded along with other host studies, and data incomplete literature according to the exclusion criteria we formulated. Finally, we extracted the number of swine with PEDV infection from the obtained studies and provided information that permitted us to estimate the prevalence of PEDV infection in pigs in mainland China. RESULTS: A total of 45 studies (including data from 15,990 pigs) met our evaluation criteria. In China, the overall estimated prevalence of PEDV infection in pigs was 44% (7113/15,990), while the estimated prevalence of PEDV infection in pigs from northern China was 37% (793/2136), lower than those in other regions of China. The prevalence of PEDV infection was associated with sampling season, category of pigs and clinical signs (diarrhea) in pigs. However, the prevalence of PEDV among pigs in China was not significantly associated with the effect of detected target genes, nor was it associated with date of study publication. CONCLUSION: Our findings suggest that PEDV infection is common among pigs in China. It is therefore necessary to carry out further research and monitor the prevalence of PEDV infection. Furthermore, powerful and effective regulatory measures should be taken in order to prevent the transmission and spread of PEDV among pig populations.

Research on the Algorithm Model for Measuring Ocean Waves Based on Satellite GPS Signals in China.

In recent years, the GPS wave buoy has been developed for in situ wave monitoring based on satellite GPS signals. Many research works have been completed on the GPS-based wave measurement technology and great progress has been achieved. The basic principle of the GPS wave buoy is to calculate the movement velocity of the buoy using the Doppler frequency shift of satellite GPS signals, and then to calculate the wave parameters from the movement velocity according to ocean wave theory. The shortage of the GPS wave buoy is the occasional occurrence of some unusual values in the movement velocity. This is mainly due to the fact that the GPS antenna is occasionally covered by sea water and cannot normally receive high-quality satellite GPS signals. The traditional solution is to remove these unusual movement velocity values from the records, which requires furthering extend the acquisition time of satellite GPS signals to ensure there is a large enough quantity of effective movement velocity values. Based on the traditional GPS wave measurement technology, this paper presents the algorithmic flow and proposes two improvement measures. On the one hand, the neural network algorithm is used to correct the unusual movement velocity data so that extending the acquisition time of satellite GPS signals is not necessary and battery power is saved. On the other hand, the Gaussian low-pass filter is used to correct the raw directional wave spectrum, which can further eliminate the influence of noise spectrum energy and improve the measurement accuracy. The on-site sea test of the SBF7-1A GPS wave buoy, developed by the National Ocean Technology Center in China, and the gravity-acceleration-type DWR-MKIII Waverider buoy are highlighted in this article. The wave data acquired by the two buoys are analyzed and processed. It can be seen from the processed results that the ocean wave parameters from the two kinds of wave buoys, such as wave height, wave period, wave direction, wave frequency spectrum, and directional wave spectrum, are in good consistency, indicating that the SBF7-1A GPS wave buoy is comparable to the traditional gravity-acceleration-type wave buoy in terms of its accuracy. Therefore, the feasibility and validity of the two improvement measures proposed in this paper are confirmed.

LPS-induced reduction of triglyceride synthesis and secretion in dairy cow mammary epithelial cells via decreased SREBP1 expression and activity.

Sterol regulatory element binding protein 1 (SREBP1) has a central regulatory effect on milk fat synthesis. Lipopolysaccharides (LPS) can induce mastitis and cause milk fat depression in cows. SREBP1 is also known to be associated with inflammatory regulation. Thus, in the current study, we hypothesized that LPS-induced milk fat depression in dairy cow mammary epithelial cells (DCMECs) operates via decreased SREBP1 expression and activity. To examine the hypothesis, DCMECs were isolated and purified from dairy cow mammary tissue and treated with LPS (10 µg/ml). LPS treatment of DCMECs suppressed lipid-metabolism-related transcription factor SREBP1 mRNA expression, nuclear translocation and protein expression, leading to reduced triglyceride content. The transcription levels of acetyl-CoA carboxylase-1 and fatty acid synthetase were significantly down-regulated in DCMECs after LPS treatment, suggesting that acetyl-CoA carboxylase-1 and fatty acid synthetase involved in de novo milk fat synthesis was regulated by SREBP1. In summary, these results suggest that LPS induces milk fat depression in dairy cow mammary epithelial cells via decreased expression of SREBP1 in a time-dependent manner.

Isolation and identification of BRV G6P[1] strain in Heilongjiang province, Northeast China.

Bovine rotavirus (BRV) is the main cause of acute gastroenteritis in calves, resulting in significant economic losses to the cattle industry worldwide. Additionally, BRV has multiple genotypes, which could enable cross-species transmission, thereby posing a significant risk to public health. However, there is a problem of multiple genotypes coexisting in BRV, and the cross-protection effect between different genotypes of rotavirus strains is not effective enough. Therefore, mastering clinical epidemic genotypes and using epidemic genotype strains for vaccine preparation is an effective means of preventing and controlling BRV. In this study, BRV strain DQ2020 in MA104 cells was identified by transmission electron microscopy (TEM), reverse transcription polymerase chain reaction (RT-PCR), and colloidal gold immunochromatographic test strips. The whole genome of BRV strain DQ2020 was sequenced and pathogenicity in suckling mice was assessed. The results showed that after 10 passages in MA104 cells, BRV strain DQ2020 induced cytopathic effects. Wheel-shaped virus particles (diameter, ~80 nm) were observed by TEM. A target band of 382 bp was detected by RT-PCR, a positive band was detected with the colloidal gold immunochromatographic test strips, and significant green fluorescence was observed by indirect immunofluorescence (IFA). The highest median tissue culture infectious dose of strain DQ2020 after 9 passages in MA104 cells was 10-4.81 viral particles/0.1 mL. Based on phylogenetic analysis of 11 gene fragments, the genotype of BRV strain DQ2020 was G6-P[1]-I2-R2-C2-M2-A11-N2-T6-E2-H3, confirming transmission of the G6-P[1] genotype in Chinese cattle herds. Further analysis showed that the isolated strain was a reassortant of bovine (VP7, VP6, NSP3, and NSP5), human (VP4, VP1, VP2, VP3, NSP2, and NSP4), and ovine (NSP1) rotaviruses. BRV strain DQ2020 caused damage to the intestinal villi of suckling mice and diarrhea, confirming pathogenicity. In summary, this study identified a reassortant strain of bovine, human, and ovine rotavirus that is pathogenic to lactating mice, and conducted whole genome sequence analysis, providing valuable insights for the genetic evolution of the virus and the development of vaccines.

A Mini-Review on the Common Antiviral Drug Targets of Coronavirus.

Coronaviruses in general are a zoonotic pathogen with significant cross-species transmission. They are widely distributed in nature and have recently become a major threat to global public health. Vaccines are the preferred strategy for the prevention of coronaviruses. However, the rapid rate of virus mutation, large number of prevalent strains, and lag in vaccine development contribute to the continuing frequent occurrence of coronavirus diseases. There is an urgent need for new antiviral strategies to address coronavirus infections effectively. Antiviral drugs are important in the prevention and control of viral diseases. Members of the genus coronavirus are highly similar in life-cycle processes such as viral invasion and replication. These, together with the high degree of similarity in the protein sequences and structures of viruses in the same genus, provide common targets for antiviral drug screening of coronaviruses and have led to important advances in recent years. In this review, we summarize the pathogenic mechanisms of coronavirus, common drugs targeting coronavirus entry into host cells, and common drug targets against coronaviruses based on biosynthesis and on viral assembly and release. We also describe the common targets of antiviral drugs against coronaviruses and the progress of antiviral drug research. Our aim is to provide a theoretical basis for the development of antiviral drugs and to accelerate the development and utilization of commonly used antiviral drugs in China.

Construction and immune effect evaluation of the S protein heptad repeat-based nanoparticle vaccine against porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV), a highly virulent enteropathogenic coronavirus, is a significant threat to the pig industry. High frequency mutations in the PEDV genome have limited the effectiveness of current vaccines in providing immune protection. Developing efficient vaccines that can quickly adapt to mutant strains is a challenging but crucial task. In this study, we chose the pivotal protein heptad repeat (HR) responsible for coronavirus entry into host cells, as the vaccine antigen. HR-Fer nanoparticles prepared using ferritin were evaluated them as PEDV vaccine candidates. Nanoparticle vaccines elicited stronger neutralizing antibody responses in mice compared to monomer vaccines. Additionally, HR protein delivered via nanoparticles increased antigen uptake by antigen-presenting cells in vitro by 2.75-fold. The collective results suggest that HR can be used as antigens for vaccines, and the HR vaccine based on ferritin nanoparticles significantly enhances immunogenicity.

Isolation and characterization of a novel S1-gene insertion porcine epidemic diarrhea virus with low pathogenicity in newborn piglets.

Porcine epidemic diarrhea virus (PEDV) causes diarrhea and vomiting in piglets, leading to a mortality rate of 100%. Due to the high frequency of mutation, it is important to monitor the evolution of PEDV and develop potential vaccine candidates. In this study, two PEDV strains (ZJ2022 and ZQ2022) were identified by PCR. These strains were subsequently isolated, and their genome sequences, growth characteristics, and pathogenicity were compared. Phylogenetic and recombination analyses revealed that both strains belonged to GIIa-subgroup, and ZQ2022 was identified as a recombinant strain derived from ZJ2022. Further sequence analysis showed that the ZJ2022 strain had a modified top region of the S1 protein due to a three amino acid insertion (T380_Y380insGGE) in the S1 gene. According to the virus growth curve, ZJ2022 exhibited better cellular adaptation than ZQ2022, with higher viral titers from 8 hpi to 24 hpi. Additionally, ZQ2022 exhibited a high level of pathogenicity, causing severe diarrhea in piglets at 36 hpi and a 100% mortality rate by 96 hpi. In contrast, ZJ2022 showed lower pathogenicity, inducing severe diarrhea in piglets at 60 hpi, with a mortality rate of 60% at 96 hpi and 100% at 120 hpi. In summary, our findings provided evidence of the undergoing mutations in Chinese PEDV strains. Furthermore, the S gene insertion strain ZJ2022 exhibited strong cellular adaptability and low pathogenicity, making it a potential candidate strain for vaccine development.

Combination of S1-N-Terminal and S1-C-Terminal Domain Antigens Targeting Double Receptor-Binding Domains Bolsters Protective Immunity of a Nanoparticle Vaccine against Porcine Epidemic Diarrhea Virus.

Variants of coronavirus porcine epidemic diarrhea virus (PEDV) frequently emerge, causing an incomplete match between the vaccine and variant strains, which affects vaccine efficacy. Designing vaccines with rapidly replaceable antigens and high efficacy is a promising strategy for the prevention of infection with PEDV variant strains. In our study, three different types of self-assembled nanoparticles (nps) targeting receptor-binding N-terminal domain (NTD) and C-terminal domain (CTD) of S1 protein, named NTDnps, CTDnps, and NTD/CTDnps, were constructed and evaluated as vaccine candidates against PEDV. NTDnps and CTDnps vaccines mediated significantly higher neutralizing antibody (NAb) titers than NTD and CTD recombinant proteins in mice. The NTD/CTDnps in varying ratios elicited significantly higher NAb titers when compared with NTDnps and CTDnps alone. The NTD/CTDnps (3:1) elicited NAb with titers up to 92.92% of those induced by the commercial vaccine. Piglets immunized with NTD/CTDnps (3:1) achieved a passive immune protection rate of 83.33% of that induced by the commercial vaccine. NTD/CTDnps (3:1) enhanced the capacity of mononuclear macrophages and dendritic cells to take up and present antigens by activating major histocompatibility complex I and II molecules to stimulate humoral and cellular immunity. These data reveal that a combination of S1-NTD and S1-CTD antigens targeting double receptor-binding domains strengthens the protective immunity of nanoparticle vaccines against PEDV. Our findings will provide a promising vaccine candidate against PEDV.

Natural hyperoside extracted from hawthorn exhibits antiviral activity against porcine epidemic diarrhea virus in vitro and in vivo.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and death in piglets, resulting in significant economic losses for the pork industry. There is an urgent need for new treatment strategies. Here, we focused on optimizing the process of purifying natural hyperoside (nHYP) from hawthorn and evaluating its effectiveness against PEDV both in vitro and in vivo. Our findings demonstrated that nHYP with a purity >98% was successfully isolated from hawthorn with an extraction rate of 0.42 mg/g. Furthermore, nHYP exhibited strong inhibitory effects on PEDV replication in cells, with a selection index of 9.72. nHYP significantly reduced the viral load in the intestines of piglets and protected three of four piglets from death caused by PEDV infection. Mechanistically, nHYP could intervene in the interaction of PEDV N protein and p53. The findings implicate nHYP as having promising therapeutic potential for combating PEDV infections.

A survey of fecal virome and bacterial community of the diarrhea-affected cattle in northeast China reveals novel disease-associated ecological risk factors.

Limited information on the virome and bacterial community hampers our ability to discern systemic ecological risk factors that cause cattle diarrhea, which has become a pressing issue in the control of disease. A total of 110 viruses, 1,011 bacterial genera, and 322 complete viral genomes were identified from 70 sequencing samples mixed with 1,120 fecal samples from 58 farms in northeast China. For the diarrheic samples, the identified virome and bacterial community varied in terms of composition, abundance, diversity, and geographic distribution in relation to different disease-associated ecological factors; the abundance of identified viruses and bacteria was significantly correlated with the host factors of clinical status, cattle type, and age, and with environmental factors such as aquaculture model and geographical location (P < 0.05); a significant interaction occurred between viruses and viruses, bacteria and bacteria, as well as between bacteria and viruses (P < 0.05). The abundance of SMB53, Butyrivibrio, Facklamia, Trichococcus, and Turicibacter was significantly correlated with the health status of cattle (P < 0.05). The proportion of BRV, BCoV, BKV, BToV, BoNoV, BoNeV, BoAstV, BEV, BoPV, and BVDV in 1,120 fecal samples varied from 1.61% to 12.05%. A series of significant correlations were observed between the prevalence of individual viruses and the disease-associated ecological factors. A genome-based phylogenetic analysis revealed high variability of 10 bovine enteric viruses. The bovine hungarovirus was initially identified in both dairy and beef cattle in China. This study elucidates the fecal virome and bacterial community signatures of cattle affected by diarrhea, and reveals novel disease-associated ecological risk factors, including cattle type, cattle age, aquaculture model, and geographical location.IMPORTANCEThe lack of data on the virome and bacterial community restricts our capability to recognize ecological risk factors for bovine diarrhea disease, thereby hindering our overall comprehension of the disease's cause. In this study, we found that, for the diarrheal samples, the identified virome and bacterial community varied in terms of composition, abundance, diversity, configuration, and geographic distribution in relation to different disease-associated ecological factors. A series of significant correlations were observed between the prevalence of individual viruses and the disease-associated ecological factors. Our study aims to uncover novel ecological risk factors of bovine diarrheal disease by examining the pathogenic microorganism-host-environment disease ecology, thereby providing a new perspective on the control of bovine diarrheal diseases.

Establishment and evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type canine distemper virus from vaccine strains.

This study sought to establish a real-time reverse transcription (RT)-PCR method to differentially detect canine distemper virus (CDV) wild-type and vaccine strains. To this end, a pair of CDV universal primers and two specific minor groove binder (MGB) probes, harboring a T/C substitution in the hemagglutinin (H) gene, were designed. Using a recombinant plasmid expressing the H gene of the CDV wild-type or vaccine strain as standards, a sensitive and specific multiplex real-time RT-PCR was established for quantitative and differential detection of CDV wild-type and vaccine strains. The limit of detection for this multiplex assay was 22.5 copies/µL and 2.98 copies/µL of viral RNA for wild-type and vaccine strains, respectively. Importantly, the wild-type and vaccine MGB probes specifically hybridized different genotypes of wild-type CDV circulating in China as well as globally administered vaccine viruses, respectively, with no cross-reactivity observed with non-CDV viruses. Moreover, this method was successfully applied for the quantitative detection of CDV RNA in tissue samples of experimentally infected breeding foxes, raccoon dogs, and minks. Additionally, the multiplex real-time RT-PCR was able to detect the viral RNA in the whole blood samples as early as 3 days post-infection, 3 to 4 days prior to the onset of clinical signs in these CDV infection animals. Hence, the established multiplex real-time RT-PCR method is useful for differentiating wild-type CDV and vaccine strains in China, and for conducting canine distemper early diagnosis as well as dynamic mechanism of CDV replication studies in vivo.

DNA Vaccine Co-Expressing Hemagglutinin and IFN-γ Provides Partial Protection to Ferrets against Lethal Challenge with Canine Distemper Virus.

Canine distemper (CD), caused by canine distemper virus (CDV), is a highly contagious and lethal disease in domestic and wild carnivores. Although CDV live-attenuated vaccines have reduced the incidence of CD worldwide, low levels of protection are achieved in the presence of maternal antibodies in juvenile animals. Moreover, live-attenuated CDV vaccines may retain residual virulence in highly susceptible species and cause disease. Here, we generated several CDV DNA vaccine candidates based on the biscistronic vector (pIRES) co-expressing virus wild-type or codon-optimized hemagglutinin (H) and nucleocapsid (N) or ferret interferon (IFN)-γ, as a molecular adjuvant, respectively. Apparently, ferret (Mustela putorius furo)-specific codon optimization increased the expression of CDV H and N proteins. A ferret model of CDV was used to evaluate the protective immune response of the DNA vaccines. The results of the vaccinated ferrets showed that the DNA vaccine co-expressing the genes of codon-optimized H and ferret IFN-γ (poptiH-IRES-IFN) elicited the highest anti-CDV serum-neutralizing antibodies titer (1:14) and cytokine responses (upregulated TNF-α, IL-4, IL-2, and IFN-γ expression) after the third immunization. Following vaccination, the animals were challenged with a lethal CDV 5804Pe/H strain with a dose of 105.0 TCID50. Protective immune responses induced by the DNA vaccine alleviated clinical symptoms and pathological changes in CDV-infected ferrets. However, it cannot completely prevent virus replication and viremia in vivo as well as virus shedding due to the limited neutralizing antibody level, which eventually contributed to a survival rate of 75% (3/4) against CDV infection. Therefore, the improved strategies for the present DNA vaccines should be taken into consideration to develop more protective immunity, which includes increasing antigen expression or alternative delivery routes, such as gene gun injection.

Octyl gallate targeting the 3C-like protease exhibits highly efficient antiviral activity against swine enteric coronavirus PEDV.

Infection with porcine epidemic diarrhea virus (PEDV) causes severe watery diarrhea in newborn piglets, leading to substantial financial losses for the swine industry. In this study, we screened small molecule drugs targeting 3 C-like protease (3CLpro) by molecular docking, and further evaluated the antiviral activity of the screened drugs against PEDV. Results showed that octyl gallate (OG), a widely used food additive, exhibited strong binding affinity with the 3CLpro active sites of PEDV. Bio-layer interferometry and fluorescence resonance energy transfer revealed that OG directly interacts with PEDV 3CLpro (KD = 549 nM) and inhibits 3CLpro activity (IC50 = 22.15 µM). OG showed a strong inhibition of PEDV replication in vitro. Virus titers were decreased by 0.58 and 0.71 log10 TCID50/mL for the CV777 and HM2017 strains, respectively. In vivo, all piglets in the PEDV-infected group died at 48 h post-infection (hpi), while 75% of piglets in the OG treatment group showed significant relief from the clinical symptoms, pathological damage, and viral loads in the jejunum and ileum. Moreover, the western blotting results showed that OG also has strong antiviral activity against other swine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). Our findings revealed that OG could be developed as a novel antiviral drug against PEDV. The OG exhibited a potential broad-spectrum antiviral drug for control of other swine enteric coronaviruses.

Molecular characterization of a rare G9P[23] porcine rotavirus isolate from China.

The fifth most important G genotype, G9 rotavirus, is recognized as an emerging genotype that is spreading around the world. Sequence analysis was completed of a rare group A rotavirus, strain G9P[23], that was designated rotavirus A pig/China/NMTL/2008/G9P[23] and abbreviated as NMTL. It was isolated from a piglet with diarrhea in China. Nucleotide sequence analysis revealed that the VP7 gene clustered within the G9 lineage VId. The VP4 gene clustered within the rare P[23] genotype. NMTL is the first porcine G9 stain reported in China. Thus, to further characterize the evolutionary diversity of the NMTL strain, all gene segments were used to draw a phylogenetic tree. Based on the new classification system of rotaviruses, the NMTL sequence revealed a G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1 genotype with close similarity to human Wa-like and porcine strains. The results showed that (i) NSP2 and NSP4 genes of NMTL exhibited higher genetic relatedness to human group A rotaviruses than to porcine strains, (ii) the VP2 and VP4 genes clustered with porcine and porcine-like human strains, and (iii) VP1 genes clustered apart from the Wa-like human and porcine clusters. In view of rotavirus evolution, this report provides additional evidence to support the notion that the human and porcine rotavirus genomes might be related.