Analysis of the recombination and evolution of the new type mutant pseudorabies virus XJ5 in China.

Pseudorabies have caused enormous economic losses in China's pig industry and have recurred on many large pig farms since late 2011. The disease is caused by highly pathogenic, antigenic variant pseudorabies virus (vPRV) strains. Our laboratory isolated a pseudorabies virus in 2015 and named it XJ5. The pathogenic ability of this mutant strain was much stronger than that of the original isolate. After we sequenced its whole genome (GenBank accession number: OP512542), we found that its overall structure was not greatly changed compared with that of the previous strain Ea (KX423960.1). The whole genome alignment showed that XJ5 had a strong genetic relationship with the strains isolated in China after 2012 reported in GenBank. Based on the isolation time of XJ5 and the mutation and recombination analysis of programs, we found that the whole genome homology of XJ5 and other strains with Chinese isolates was greater than 95%, while the homology with strains outside Asia was less than 94%, which indicated that there may be some recombination and mutation patterns. We found that virulent PRV isolates emerged successively in China in 2011 and formed two different evolutionary clades from foreign isolates. At the same time, this may be due to improper immunization and the presence of wild strains in the field, and recent reports have confirmed that Bartha vaccine strains recombine with wild strains to obtain new pathogenic strains. We performed genetic evolution analysis of XJ5 isolated and sequenced in our laboratory to trace its possible mutations and recombination. We found that XJ5 may be the result of natural mutation of a virus in a branch of mutant strains widely existing in China.

Genetic characterization and phylogenetic analysis of the S genes of porcine epidemic diarrhea virus isolates from China from 2020 to 2023.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that has been the main cause of diarrhea in piglets since 2010 in China. The aim of this study was to investigate sequence variation and recombination events in the spike (S) gene of PEDV isolates from China. Thirty complete S gene sequences were obtained from PEDV-positive samples collected in six provinces in China from 2020 to 2023. Phylogenetic analysis showed that 10% (3/30) belonged to subtype GII-a, 6.67% (2/30) were categorized as subtype GII-b, 66.67% (20/30) were categorized as subtype GII-c, and 16.66% (5/30) were clustered with the S-INDEL strains. Amino acid sequence alignments showed that, when compared to strains of other subtypes, the GII-c strains had two characteristic amino acid substitutions (N139D and I289M). Five S-INDEL subtype strains had a single amino acid deletion (139N) and four amino acid substitutions (N118G, T137S, A138S, and D141G). Recombination analysis allowed six putative recombination events to be identified, one involving recombination between GII-c strains, two involving GII-c and GII-b strains, two involving GII-c and GI-a strains, and one involving GII-a and GI-b strains. These results suggest that recombination between PEDV strains has been common and complex in recent years and is one of the main reasons for the continuous variation of PEDV strains.

Development of a colloidal gold immunochromatographic strip for the simultaneous detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus.

In recent years, porcine diarrhea-associated viruses have caused significant economic losses globally. These viruses present similar clinical symptoms, such as watery diarrhea, dehydration, and vomiting. Co-infections with porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are common. For the rapid and on-site preliminary diagnosis on the pig farms, this study aimed to develop a colloidal gold immunochromatography assay (GICA) strip for the detection of PEDV and TGEV simultaneously. The GICA kit showed that there was no cross-reactivity with the other five common porcine viruses. With visual observation, the lower limits were approximately 104 TCID50/mL and 104 TCID50/mL for PEDV and TGEV, respectively. The GICA strip could be stored at 4°C or 25°C for 12 months without affecting its efficacy. To validate the GICA strip, 121 clinical samples were tested. The positive rates of PEDV and TGEV were 42.9 and 9.9%, respectively, and the co-infection rate of the two viruses was 5.8% based on the duplex GICA strip. Thus, the established GICA strip is a rapid, specific, and stable tool for on-site preliminary diagnosis of PEDV- and TGEV-associated diarrhea.

A novel virus-like particles vaccine induces broad immune protective against deltacoronavirus in piglets.

Coronaviruses (CoVs) comprise a group of important human and animal pathogens that threaten public health because of their interspecies transmission potential to humans. However, virus-like particles (VLPs) constitute versatile tools in CoVs vaccine development due to their favorable immunological characteristics. Here, we engineered the VLPs composed of the spike (S), membrane (M), and envelope (E) structural proteins of the Porcine deltacoronavirus (PDCoV) and examined their immune responses in mice. Neutralization assays and flow Cytometry demonstrated that PDCoV VLPs induced highly robust neutralizing antibodies (NAbs) and elicited cellular immunity. To assess the protective efficacy of VLPs in newborn piglets, pregnant sows received vaccinations with either a PDCoV-inactivated vaccine or VLPs at 40 and 20 days before delivery. Five days post-farrowing, piglets were orally challenged with the PDCoV strain. Severe diarrhea, high viral RNA copies, and substantial intestinal villus atrophy were detected in piglets born to unimmunized sows. However, piglets from sows immunized with VLPs exhibited high NAbs titers and markedly reduced microscopic damage to the intestinal tissues, with no piglet showing diarrhea. Hence, the results indicate that the VLPs are a potential clinical candidate for PDCoV vaccination, while the strategy may serve as a platform for developing other coronavirus vaccines.

The synergy of recombinant NSP4 and VP4 from porcine rotavirus elicited a strong mucosal response.

Porcine rotavirus (PoRV) is one of the main pathogens causing diarrhea in piglets, and multiple genotypes coexist. However, an effective vaccine is currently lacking. Here, the potential adjuvant of nonstructural protein 4 (NSP4) and highly immunogenic structural protein VP4 prompted us to construct recombinant NSP486-175aa (NSP4*) and VP426-476aa (VP4*) proteins, combine them as immunogens to evaluate their efficacy. Results indicated that NSP4* enhanced systemic and local mucosal responses induced by VP4*. The VP4*-IgG, VP4*-IgA in feces and IgA-secreting cells in intestines induced by the co-immunization were significantly higher than those induced by VP4* alone. Co-immunization of NSP4* and VP4* also induced strong cellular immunity with significantly increased IFN-λ than the single VP4*. Summarily, the NSP4* as a synergistical antigen exerted limited effects on the PoRV NAbs elevation, but conferred strong VP4*-specific mucosal and cellular efficacy, which lays the foundation for the development of a more effective porcine rotavirus subunit vaccine.

Recombinant bivalent subunit vaccine combining truncated VP4 from P[7] and P[23] induces protective immunity against prevalent porcine rotaviruses.

Porcine rotaviruses (PoRVs) cause severe economic losses in the swine industry. P[7] and P[23] are the predominant genotypes circulating on farms, but no vaccine is yet available. Here, we developed a bivalent subunit PoRV vaccine using truncated versions (VP4*) of the VP4 proteins from P[7] and P[23]. The vaccination of mice with the bivalent subunit vaccine elicited more robust neutralizing antibodies (NAbs) and cellular immune responses than its components, even at high doses. The bivalent subunit vaccine and inactivated bivalent vaccine prepared from strains PoRVs G9P[7] and G9P[23] were used to examine their protective efficacy in sows and suckling piglets after passive immunization. The immunized sows showed significantly elevated NAbs in the serum and colostrum, and the suckling piglets acquired high levels of sIgA antibodies from the colostrum. Challenging subunit-vaccinated or inactivated-vaccinated piglets with homologous virulent strains did not induce diarrhea, except in one or two piglets, which had mild diarrhea. Immunization with the bivalent subunit vaccine and inactivated vaccine also alleviated the microscopic lesions in the intestinal tissues caused by the challenge with the corresponding homologous virulent strain. However, all the piglets in the challenged group displayed mild to watery diarrhea and high levels of viral shedding, whereas the feces and intestines of the piglets in the bivalent subunit vaccine and inactivated vaccine groups had lower viral loads. In summary, our data show for the first time that a bivalent subunit vaccine combining VP4*P[7] and VP4*P[23] effectively protects piglets against the diarrhea caused by homologous virulent strains.IMPORTANCEPoRVs are the main causes of diarrhea in piglets worldwide. The multisegmented genome of PoRVs allows the reassortment of VP4 and VP7 genes from different RV species and strains. The P[7] and P[23] are the predominant genotypes circulating in pig farms, but no vaccine is available at present in China. Subunit vaccines, as nonreplicating vaccines, are an option to cope with variable genotypes. Here, we have developed a bivalent subunit candidate vaccine based on a truncated VP4 protein, which induced robust humoral and cellular immune responses and protected piglets against challenge with homologous PoRV. It also appears to be safe. These data show that the truncated VP4-protein-based subunit vaccine is a promising candidate for the prevention of PoRV diarrhea.

A Novel Polymer Nanoparticle Polydimethyl Diallyl Ammonium Chloride as An Adjuvant Enhances the Immune Response of SARS-CoV-2 Subunit Vaccine.

The Coronavirus Disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has a significant impact on global health and the economy. It has underscored the urgent need for a stable, easily produced and effective vaccine. This study presents a novel approach using SARS-CoV-2 spike (S) protein-conjugated nanoparticles (NPs) in combination with cyclic GMP-AMP (cGAMP) (S-NPs-cGAMP) as a subunit vaccine. When mice are immunized, the antiserum of S-NPs-cGAMP group exhibits a 16-fold increase in neutralizing activity against a pseudovirus, compared to S protein group. Additionally, S-NPs-cGAMP induces even higher levels of neutralizing antibodies. Remarkably, the vaccine also triggers a robust humoral immune response, as evidenced by a notable elevation in virus-specific IgG and IgM antibodies. Furthermore, after 42 days of immunization, there is an observed increase in specific immune cell populations in the spleen. CD3+CD4+ and CD3+CD8+T lymphocytes, as well as B220+CD19+ and CD3-CD49b+ NK lymphocytes, show an upward trend, indicating a positive cellular immune response. Moreover, the S-NPs-cGAMP demonstrates promising results against the Delta strain and exhibits good cross-neutralization potential against other variants. These findings suggest that pDMDAAC NPs is potential adjuvant and could serve as a versatile platform for future vaccine development.

Lipidomics reveals the significance and mechanism of the cellular ceramide metabolism for rotavirus replication.

As one of the most important causative agents of severe gastroenteritis in children, piglets, and other young animals, species A rotaviruses have adversely impacted both human health and the global swine industry. Vaccines against rotaviruses (RVs) are insufficiently effective, and no specific treatment is available. To understand the relationships between porcine RV (PoRV) infection and enterocytes in terms of the cellular lipid metabolism, we performed an untargeted liquid chromatography mass spectrometry (LC-MS) lipidomics analysis of PoRV-infected IPEC-J2 cells. Herein, a total of 451 lipids (263 upregulated lipids and 188 downregulated lipids), spanning sphingolipid, glycerolipid, and glycerophospholipids, were significantly altered compared with the mock-infected group. Interestingly, almost all the ceramides among these lipids were upregulated during PoRV infection. LC-MS analysis was used to validated the lipidomics data and demonstrated that PoRV replication increased the levels of long-chain ceramides (C16-ceramide, C18-ceramide, and C24-ceramide) in cells. Furthermore, we found that these long-chain ceramides markedly inhibited PoRV infection and that their antiviral actions were exerted in the replication stage of PoRV infection. Moreover, downregulation of endogenous ceramides with the ceramide metabolic inhibitors enhanced PoRV propagation. Increasing the levels of ceramides by the addition of C6-ceramide strikingly suppressed the replication of diverse RV strains. We further found that the treatment with an apoptotic inhibitor could reverse the antiviral activity of ceramide against PoRV replication, demonstrating that ceramide restricted RV infection by inducing apoptosis. Altogether, this study revealed that ceramides played an antiviral role against RV infection, providing potential approaches for the development of antiviral therapies.IMPORTANCERotaviruses (RVs) are among the most important zoonosis viruses, which mainly infected enterocytes of the intestinal epithelium causing diarrhea in children and the young of many mammalian and avian species. Lipids play an essential role in viral infection. A comprehensive understanding of the interaction between RV and lipid metabolism in the enterocytes will be helpful to control RV infection. Here, we mapped changes in enterocyte lipids following porcine RV (PoRV) infection using an untargeted lipidomics approach. We found that PoRV infection altered the metabolism of various lipid species, especially ceramides (derivatives of the sphingosine). We further demonstrated that PoRV infection increased the accumulation of ceramides and that ceramides exerted antiviral effects on RV replication by inducing apoptosis. Our findings fill a gap in understanding the alterations of lipid metabolism in RV-infected enterocytes and highlight the antiviral effects of ceramides on RV infection, suggesting potential approaches to control RV infection.

Isolation and characterization of a G9P[23] porcine rotavirus strain AHFY2022 in China.

Rotavirus group A (RVA) is a main pathogen causing diarrheal diseases in humans and animals. Various genotypes are prevalent in the Chinese pig herd. The genetic diversity of RVA lead to distinctly characteristics. In the present study, a porcine RVA strain, named AHFY2022, was successfully isolated from the small intestine tissue of piglets with severe diarrhea. The AHFY2022 strain was identified by cytopathic effects (CPE) observation, indirect immunofluorescence assay (IFA), electron microscopy (EM), high-throughput sequencing, and pathogenesis to piglets. The genomic investigation using NGS data revealed that AHFY2022 exhibited the genotypes G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1, using the online platform the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) (https://www.bv-brc.org/). Moreover, experimental inoculation in 5-day-old and 27-day-old piglets demonstrated that AHFY2022 caused severe diarrhea, fecal shedding, small intestinal villi damage, and colonization in all challenged piglets. Taken together, our results detailed the virological features of the porcine rotavirus G9P[23] from China, including the whole-genome sequences, genotypes, growth kinetics in MA104 cells and the pathogenicity in suckling piglets.

A novel recombinant S-based subunit vaccine induces protective immunity against porcine deltacoronavirus challenge in piglets.

IMPORTANCE: As an emerging porcine enteropathogenic coronavirus that has the potential to infect humans, porcine deltacoronavirus (PDCoV) is receiving increasing attention. However, no effective commercially available vaccines against this virus are available. In this work, we designed a spike (S) protein and receptor-binding domain (RBD) trimer as a candidate PDCoV subunit vaccine. We demonstrated that S protein induced more robust humoral and cellular immune responses than the RBD trimer in mice. Furthermore, the protective efficacy of the S protein was compared with that of inactivated PDCoV vaccines in piglets and sows. Of note, the immunized piglets and suckling pig showed a high level of NAbs and were associated with reduced virus shedding and mild diarrhea, and the high level of NAbs was maintained for at least 4 months. Importantly, we demonstrated that S protein-based subunit vaccines conferred significant protection against PDCoV infection.

Genetic signatures associated with the virulence of porcine epidemic diarrhea virus AH2012/12.

IMPORTANCE: Porcine epidemic diarrhea (PED) caused by PED virus (PEDV) remains a big threat to the swine industry worldwide. Vaccination with live attenuated vaccine is a promising method to prevent and control PED, because it can elicit a more protective immunity than the killed vaccine, subunit vaccine, and so on. In this study, we found two obvious deletions in the genome of a high passage of AH2012/12. We further confirmed the second deletion which contains seven amino acids at the carboxy-terminus of the S2 gene and the start codon of ORF3 can reduce its pathogenicity in vivo. Animal experiments indicated that the recombinant PEDV with deleted carboxy-terminus of S gene showed higher IgG, IgA, neutralization antibodies, and protection effects against virus challenge than the killed vaccine. These data reveal that the engineering of the carboxy-terminus of the S2 gene may be a promising method to develop live attenuated vaccine candidates of PEDV.

Lysine-Specific Demethylase 1 Promises to Be a Novel Target in Cancer Drug Resistance: Therapeutic Implications.

Chemotherapy, targeted therapy, and immunotherapy are effective against most tumors, but drug resistance remains a barrier to successful treatment. Lysine-specific demethylase 1 (LSD1), a member of histone demethylation modifications, can regulate invasion, metastasis, apoptosis, and immune escape of tumor cells, which are associated with tumorigenesis and tumor progression. Recent studies suggest that LSD1 ablation regulates resensitivity of tumor cells to anticarcinogens containing immune checkpoint inhibitors (ICIs) via multiple upstream and downstream pathways. In this review, we describe the recent findings about LSD1 biology and its role in the development and progression of cancer drug resistance. Further, we summarize LSD1 inhibitors that have a reversal or resensitive effect on drug resistance and discuss the possibility of targeting LSD1 in combination with other agents to surmount resistance.

Analysis on the genome of a teschovirus type 1 isolates with swine diarrhea.

Porcine Teschoviruses (PTVs) are associated with polioencephalomyelitis and various diseases, including reproductive and gastrointestinal disorders of pigs and wild boars, but rarely detected in the feces of pigs. In this study, a sample of swine diarrhea that tested positive for PTVs is subjected to high-throughput sequencing. The viral genome was 7221 nucleotides (nt) in length, which was consisted of twelve genes. Phylogenetic analysis showed and it was closely related to the PTV-HNMY(MG755212.1). The nucleotide homology of VP1 gene of PTVs JS2021 with PTV-1AF 296102.1 reached 82.97%, belonging to a branch of PTV-1 serotype. The nucleotide homology of VP1 protein with other serotypes of PTV is quite different from that of other serotypes of PTV. Bioinformatics analysis showed that PTVs have four capsid proteins, namely VP1, VP2, VP3 and VP4. The VP1 encodes a 29 kDa protein, which is the main protective antigen, a theoretical isoelectric point of 6.73, no transmembrane domain, no signal peptide and potential phosphorylation site. The VP1 protein is an unstable hydrophilic intracellular protein, which contains four secondary structures: irregular curl (c), extended chain (e), α-helix (h) and ß-folded (t). The tertiary structure is heart-shaped and has multiple B cell epitopes. By analyzing the tertiary structure, we found that the amino acid at position 129 of VP1 mutated and reduction a larger alpha helix. This may lead to the main cause of piglet diarrhea. These findings enriched our knowledge of the viruses in the role of swine diarrhea, and help to develop an effective strategy for disease prevention and control.

Molecular epidemiological investigation of group A porcine rotavirus in East China.

Group A porcine rotavirus (RVA) is a serious threat to the breeding industry worldwide, which was associated with severe diarrhea in piglets. However, the prevalence and molecular characterizations of RVA circulating in farms of East China remains largely unknown. Five hundred and ninety-four samples were collected from 35 farms in East China from September 2017 to December 2019. The results showed that 16.8% was positive for RVA of all samples. Among different types of samples, the highest positive rate of RVA was intestinal samples (19.5%), and among pigs at different growth stages, the highest detection rate of RVA in piglets was 18.5%. Furthermore, the VP7 and VP4 genes of nine positive samples were sequenced for alignment and phylogenetic analysis. Phylogenetic analysis revealed that the nine isolates belong to four kinds of genotype combinations correspondingly: G9P[7](5/9), G5P[13](2/9), G9P[13](1/9), and G5P[7](1/9).The data suggested that multiple genotypes combinations of RVA were circulating in pigs in East China. Thus, it's necessary to continuously survey the prevalence of RVA in pigs, aiding the rational application of vaccines or other measures for the prevention and control of RVA spread.

CMPK2 is a host restriction factor that inhibits infection of multiple coronaviruses in a cell-intrinsic manner.

Coronaviruses (CoVs) comprise a group of important human and animal pathogens. Despite extensive research in the past 3 years, the host innate immune defense mechanisms against CoVs remain incompletely understood, limiting the development of effective antivirals and non-antibody-based therapeutics. Here, we performed an integrated transcriptomic analysis of porcine jejunal epithelial cells infected with porcine epidemic diarrhea virus (PEDV) and identified cytidine/uridine monophosphate kinase 2 (CMPK2) as a potential host restriction factor. CMPK2 exhibited modest antiviral activity against PEDV infection in multiple cell types. CMPK2 transcription was regulated by interferon-dependent and interferon regulatory factor 1 (IRF1)-dependent pathways post-PEDV infection. We demonstrated that 3'-deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) catalysis by Viperin, another interferon-stimulated protein, was essential for CMPK2's antiviral activity. Both the classical catalytic domain and the newly identified antiviral key domain of CMPK2 played crucial roles in this process. Together, CMPK2, viperin, and ddhCTP suppressed the replication of several other CoVs of different genera through inhibition of the RNA-dependent RNA polymerase activities. Our results revealed a previously unknown function of CMPK2 as a restriction factor for CoVs, implying that CMPK2 might be an alternative target of interfering with the viral polymerase activity.

Transcriptomic and antiviral analyses of PoIFN-Delta5 against porcine enteric viruses in porcine intestinal epithelial cells.

The interferon-delta family was first reported in domestic pigs and belongs to the type I interferon (IFN-I) family. The enteric viruses could cause diarrhea in newborn piglets with high morbidity and mortality. We researched the function of the porcine IFN-delta (PoIFN-δ) family in the porcine intestinal epithelial cells (IPEC-J2) cells infected with porcine epidemic diarrhea virus (PEDV). Our study found that all PoIFN-δs shared a typical IFN-I signature and could be divided into five branches in the phylogenic tree. Different strains of PEDV could induce typical IFN transitorily, and the virulent strain AH2012/12 had the strongest induction of porcine IFN-δ and IFN-alpha (PoIFN-α) in the early stage of infection. In addition, it was found that PoIFN-δ5/6/9/11 and PoIFN-δ1/2 were highly expressed in the intestine. PoIFN-δ5 had a better antiviral effect on PEDV compared to PoIFN-δ1 due to its higher induction of ISGs. PoIFN-δ1 and PoIFN-δ5 also activated JAK-STAT and IRS signaling. For other enteric viruses, transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus (PoRV), PoIFN-δ1 and PoIFN-δ5 both showed an excellent antiviral effect. Transcriptome analyses uncovered the differences in host responses to PoIFN-α and PoIFN-δ5 and revealed thousands of differentially expressed genes were mainly enriched in the inflammatory response, antigen processing and presentation, and other immune-related pathways. PoIFN-δ5 would be a potential antiviral drug, especially against porcine enteric viruses. These studies were the first to report the antiviral function against porcine enteric viruses and broaden the new acquaintances of this type of interferon though not novelly discovered.

Characterization and epitope mapping of monoclonal antibodies against PEDV N protein.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration and high mortality in neonatal piglets. The nucleocapsid (N) protein of PEDV is a highly conserved protein with strong immunogenicity and palys an important role in PEDV diagnosis. However, epitopes on the PEDV N protein have not yet been well characterized. Here, 32 monoclonal antibodies (mAbs) against the PEDV N protein were produced and identified. Six new epitopes were first identified by using a high-throughput epitope mapping method named AbMap. Sequence analysis revealed that among the six epitopes five epitopes were highly conserved among different PEDV strains. We also confirmed that the mAbs derived from the six epitopes of PEDV N protein, have no cross-reactivity with transmissible gastro enteritis virus or porcine delta coronavirus. These mAbs and their defined epitopes will help to understand the N protein structure and immunological characteristics, and to develop a rapid, accurate PEDV diagnosis method.

Development of a Competitive Enzyme-Linked Immunosorbent Assay Targeting the-p30 Protein for Detection of Antibodies against African Swine Fever Virus.

African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs of all breeds and ages, caused by African swine fever virus (ASFV). Due to the absence of a safe and efficacious vaccine, accurate laboratory diagnosis is critical for the control of ASF prevention. The p30 protein is immunogenic and stimulates a high level of antibody response to ASFV infection. We developed a panel of 4 monoclonal antibodies (mAbs) against p30 protein, and mAb-2B4 showed the highest percent of inhibition (PI) of 70% in the solid phase blocking ELISA (bELISA). Epitope mapping revealed the mAb-2B4 recognized the epitope of aa 12-18 of p30, which is conserved among various ASFV genotypes. Subsequently, a competitive enzyme-linked immunosorbent assay (cELISA) was established using HRP-labeled mAb-2B4. The cutoff for discrimination between 98 negative sera and 40 positive sera against ASFV was determined by plotting a receiver operating characteristic (ROC) curve. It yielded the area under the curve (AUC) of 0.998, and a diagnostic specificity of 97.96% and a sensitivity of 97.5% were achieved when the cutoff value was determined at 37.1%. Furthermore, the results showed an excellent repeatability of the established cELISA and no cross-reaction to antisera against six other pig pathogens. Additionally, the cELISA detected a titer of 1:256 in the positive standard serum. Overall, mAb-2B4 showed a conserved epitope and high ability to be inhibited by positive sera in ASFV antibody detection. The cELISA based on HRP-labeled mAb-2B4 offers an alternative to other assays for a broader diagnostic coverage of ASFV infection.

Identification of Cell Types and Transcriptome Landscapes of Porcine Epidemic Diarrhea Virus-Infected Porcine Small Intestine Using Single-Cell RNA Sequencing.

Swine coronavirus-porcine epidemic diarrhea virus (PEDV) with specific susceptibility to pigs has existed for decades, and recurrent epidemics caused by mutant strains have swept the world again since 2010. In this study, single-cell RNA sequencing was used to perform for the first time, to our knowledge, a systematic analysis of pig jejunum infected with PEDV. Pig intestinal cell types were identified by representative markers and identified a new tuft cell marker, DNAH11. Excepting enterocyte cells, the goblet and tuft cells confirmed susceptibility to PEDV. Enrichment analyses showed that PEDV infection resulted in upregulation of cell apoptosis, junctions, and the MAPK signaling pathway and downregulation of oxidative phosphorylation in intestinal epithelial cell types. The T cell differentiation and IgA production were decreased in T and B cells, respectively. Cytokine gene analyses revealed that PEDV infection downregulated CXCL8, CXCL16, and IL34 in tuft cells and upregulated IL22 in Th17 cells. Further studies found that infection of goblet cells with PEDV decreased the expression of MUC2, as well as other mucin components. Moreover, the antimicrobial peptide REG3G was obviously upregulated through the IL33-STAT3 signaling pathway in enterocyte cells in the PEDV-infected group, and REG3G inhibited the PEDV replication. Finally, enterocyte cells expressed almost all coronavirus entry factors, and PEDV infection caused significant upregulation of the coronavirus receptor ACE2 in enterocyte cells. In summary, this study systematically investigated the responses of different cell types in the jejunum of piglets after PEDV infection, which deepened the understanding of viral pathogenesis.

Comparison of pathogenicity of porcine deltacoronavirus CZ2020 from cell culture and intestinal contents in 27-day-old piglets.

Porcine deltacoronavirus (PDCoV) is an emenging swine enteropathogenic coronavirus that can cause high mortality rate. It affects pigs of all ages, but most several in neonatal piglets. Little is known regarding the pathogenicity of PDCoV against 27-day-old piglets. In this study, 27-day-old piglets were experimentally infected with PDCoV CZ2020 from cell culture, the challenged piglets do not have obvious symptoms from 1 to 7 days post-challenge (DPC), while viral shedding was detected in rectal swab at 1 DPC. Tissues of small intestines displayed slight macroscopic and microscopic lesions with no viral antigen detection. On the other hand, 27-day-old piglets were infected with PDCoV from intestinal contents, the piglets developed mild to severe diarrhea, shedding increasing from 2 to 7 DPC, and developed macroscopic and microscopic lesions in small intestines with clear viral antigen confirmed by immunohistochemistry staining. Indicating the small intestine was still the major target organ in PDCoV-challenged pigs at the age of 27-day-old. Diarrhea caused by PDCoV from intestinal contents in 27-day-old piglets is less reported. Thus, our results might provide new insights into the pathogenesis of PDCoV.