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.

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.

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.

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.

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.

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.

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.

Nonstructural Protein 1 of Variant PEDV Plays a Key Role in Escaping Replication Restriction by Complement C3.

Zoonotic coronaviruses represent an ongoing threat to public health. The classical porcine epidemic diarrhea virus (PEDV) first appeared in the early 1970s. Since 2010, outbreaks of highly virulent PEDV variants have caused great economic losses to the swine industry worldwide. However, the strategies by which PEDV variants escape host immune responses are not fully understood. Complement component 3 (C3) is considered a central component of the three complement activation pathways and plays a crucial role in preventing viral infection. In this study, we found that C3 significantly inhibited PEDV replication in vitro, and both variant and classical PEDV strains induced high levels of interleukin-1ß (IL-1ß) in Huh7 cells. However, the PEDV variant strain reduces C3 transcript and protein levels induced by IL-1ß compared with the PEDV classical strain. Examination of key molecules of the C3 transcriptional signaling pathway revealed that variant PEDV reduced C3 by inhibiting CCAAT/enhancer-binding protein ß (C/EBP-ß) phosphorylation. Mechanistically, PEDV nonstructural protein 1 (NSP1) inhibited C/EBP-ß phosphorylation via amino acid residue 50. Finally, we constructed recombinant PEDVs to verify the critical role of amino acid 50 of NSP1 in the regulation of C3 expression. In summary, we identified a novel antiviral role of C3 in inhibiting PEDV replication and the viral immune evasion strategies of PEDV variants. Our study reveals new information on PEDV-host interactions and furthers our understanding of the pathogenic mechanism of this virus. IMPORTANCE The complement system acts as a vital link between the innate and the adaptive immunity and has the ability to recognize and neutralize various pathogens. Activation of the complement system acts as a double-edged sword, as appropriate levels of activation protect against pathogenic infections, but excessive responses can provoke a dramatic inflammatory response and cause tissue damage, leading to pathological processes, which often appear in COVID-19 patients. However, how PEDV, as the most severe coronavirus causing diarrhea in piglets, regulates the complement system has not been previously reported. In this study, for the first time, we identified a novel mechanism of a PEDV variant in the suppression of C3 expression, showing that different coronaviruses and even different subtype strains differ in regulation of C3 expression. In addition, this study provides a deeper understanding of the mechanism of the PEDV variant in immune escape and enhanced virulence.

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.

A flagellin-adjuvanted inactivated porcine epidemic diarrhea virus (PEDV) vaccine provides enhanced immune protection against PEDV challenge in piglets.

Since late 2010, outbreaks of porcine epidemic diarrhea (PED) have been reported in the swine industry in China. A variant PEDV strain that differs from strain CV777 causes prevalent PEDV infections which commercial vaccines based on CV777 cannot provide complete protection. In this study, we designed a new vaccine based on the epidemic PEDV strain AH2012/12, adjuvanted with flagellin, a mucosal adjuvant that induces mucosal and systemic production of IgA. Three groups of pregnant sows were immunized twice, with a 14-day interval, with PEDV adjuvanted with flagellin, PEDV alone, or PBS before farrowing, and newborn piglets from each group were selected and challenged with PEDV. Immunization with this vaccine elicited high levels of IgG, IgA, and neutralizing antibodies in the serum and colostrum of sows, and newborn piglets were protected against PEDV while suckling. This study should guide the prevention and control strategies for PEDV infection, thereby reducing the losses associated with this virus.

Therapeutic effects of saponin components on porcine reproductive and respiratory syndrome virus-infected piglets.

The present study aimed to evaluate the potential therapeutic effects of Anemoside B4 (AB4), Panax notoginseng saponins (PNS), Notoginsenoside R1 (SR1), Saikosaponin A (SSA) and Saikosaponin D (SSD) on piglets infected with porcine reproductive and respiratory syndrome virus (PRRSV). A total of 132 completely healthy piglets were randomly divided into 22 groups consisting of six animals each. Control piglets were intramuscularly injected with 2 ml of PRRSV (NJGC strain) solution containing 106  TCID50  virus/ml. For low-, middle- and high-dose saponin treatment groups, the piglets were initially administrated with the same volume of PRRSV solution, followed by intraperitoneal injection with AB4, PNS, SR1, SSA or SSD at 1, 5 or 10 mg/kg b.w. on day 3. The piglets in drug control group were intraperitoneally injected with 10 mg/kg b.w. of each saponin without prior PRRS challenge, while those in blank control group were injected with the same amount of normal saline. The results indicated that all the five saponin components could decrease the incidence and severity of PRRSV-induced immunopathological damages, including the elevated body temperature, weight loss, anaemia and internal inflammation. Moreover, the saponin components could enhance protein absorption and immune responses. Taken together, this study reveals that the saponin components are effective against PRRSV infection and strengthen the immune system and thus may serve as potential antiviral therapeutic agents.

Efficient Sensor Placement Optimization for Shape Deformation Sensing of Antenna Structures with Fiber Bragg Grating Strain Sensors.

This paper investigates the problem of an optimal sensor placement for better shape deformation sensing of a new antenna structure with embedded or attached Fiber Bragg grating (FBG) strain sensors. In this paper, the deformation shape of the antenna structure is reconstructed using a strain⁻displacement transformation, according to the measured discrete strain data from limited FBG strain sensors. Moreover, a two-stage sensor placement method is proposed using a derived relative reconstruction error equation. In this method, the initial sensor locations are determined using the principal component analysis based on orthogonal trigonometric (i.e., QR) decomposition, and then a new location is sequentially added into the initial sensor locations one by one by minimizing the relative reconstruction error considering information redundancy. The numerical simulations are conducted, and the comparisons show that the proposed method is advantageous in terms of the sensor distribution and computational cost. Experimental validation is performed using an antenna experimental platform equipped with an optimal FBG strain sensor configuration, and the reconstruction results show good agreements with those measured directly from displacement sensors. The proposed method has a large potential for the strain sensor placement of complex structures, and the proposed antenna structure with FBG strain sensors can be applied to the future wing-skin antenna or flexible space-based antenna.

Inactivated chimeric porcine circovirus (PCV) 1-2 vaccines based on genotypes 2b and 2d exhibit similar immunological effectiveness in protecting pigs against challenge with PCV2b strain 0233.

Porcine circovirus type 2 (PCV2) is subdivided into four genotypes: PCV2a, PCV2b, PCV2c and PCV2d. Here, for the first time, we compared the efficacy of two experimental inactivated chimeric PCV1-2 vaccines based on genotypes 2b and 2d. Seventeen 3-week-old pigs were divided randomly into four groups. Group 1 and 2 pigs were inoculated with genotype 2b- and 2d-based inactivated vaccines, respectively. At 28 days post-vaccination (DPV), pigs in groups 1-3 were challenged with the PCV2b 0233 strain. All experimental pigs were necropsied at 21 days post-challenge (DPC). Pigs vaccinated with the genotype 2b- or 2d-based vaccine had high antibody titres and lower PCV2b copy numbers in samples of sera, faeces and nasal secretions compared with pigs in the unvaccinated challenge group. Interestingly, we detected no DNA from the challenge strain in the superficial inguinal lymph nodes of the pigs immunized with the PCV2b vaccine, while one pig in the PCV2d- immunized group had detectable DNA from the challenge strain at 21 DPC. We found no significant differences in the humoral immune response, PCV2b load, or PCV-related microscopic lesions between the two vaccinated groups post-challenge. Therefore, both vaccines were equally effective at inducing immunity against challenge with PCV2b strain 0233.

Comparative efficacy of experimental inactivated and live-attenuated chimeric porcine circovirus (PCV) 1-2b vaccines derived from PCV1 and PCV2b isolates originated in China.

BACKGROUND: Porcine circovirus type-2b (PCV2b) is recognized as the etiological agent of the various clinical manifestations of porcine circovirus-associated disease (PCVAD). Previous studies have demonstrated effectiveness of chimeric PCV1-2 vaccines against PCV2b challenge. In this study, the efficacy of inactivated and live-attenuated (2 × 10(3.5) or 2 × 10(4.0) 50% tissue culture infective dose [TCID50] dose) chimeric PCV1-2b vaccines was compared side-by-side in conventional pigs. METHODS: Twenty-seven non-PCV2 viremic pigs without PCV2-specific antibody were randomly divided into six groups, including four vaccinated and challenged groups, a nonvaccinated challenged group, and a mock group. All pigs except those in the mock group were challenged at 28 days post vaccination (DPV) using PCV2b. RESULTS: Both inactivated and live-attenuated chimeric PCV1-2b vaccines induced a robust antibody responses, and significantly decreased microscopic lesion and lower viral loads in serum or superficial inguinal lymph nodes (SILN) compared with that in the nonvaccinated challenged group. PCV2 antibody titers decreased after 7 days post challenge (DPC) in pigs administered the inactivated PCV1-2b vaccine and they were lower than those in pigs inoculated with live-attenuated PCV1-2b on the day of necropsy. Moreover, no viremia was present in pigs inoculated with live-attenuated PCV1-2b vaccine at 21 DPC regardless of the dose difference. CONCLUSIONS: The results demonstrated that both inactivated and live-attenuated chimeric PCV1-2b vaccines were effective to induce protective immunity against PCV2b infection.

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.

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.

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.

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.

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.

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.