Porcine reproductive and respiratory syndrome virus （PRRSV） remains a pathogen causing substantial economic losses to China’s swine industry. Porcine reproductive and respiratory syndrome virus type 2 （PRRSV-2） currently predominates in China， exhibiting a complex epidemiological pattern characterized by co-circulating multiple lineages， which significantly complicates disease control measures. This study aimed to evaluate the diagnostic potential of metatranscriptomic sequencing in discriminating PRRSV-2 mixed infections involving multiple lineages. A PRRSV-2-positive serum sample collected from a swine farm in Sichuan Province was subjected to metatranscriptomic sequencing. This approach successfully recovered complete viral genome sequences. Subsequent comprehensive analysis， including phylogenetic reconstruction （IQ-TREE）， similarity comparison （Megalign）， and recombination analysis （RDP4/SimPlot）， was employed to elucidate the evolutionary and recombination characteristics of the viruses. The results demonstrate the successful identification of three distinct PRRSV-2 strains from this single serum sample： SC01-2025 （15 023 bp）， SC02-2025 （14 954 bp）， and SC03-2025 （15 293 bp）—the phylogenetic classification assigned these strains to lineages 1.8， 3， and 8.7， respectively.SC01-2025 was identified as a recombinant between lineage 1.8 （NADC30） and lineage 5 （VR2332）. While its full genome shared 88.09% nucleotide similarity with the NADC30 strain， regions encompassing NSP3-7， NSP9-12， and ORF3 exhibited higher similarity to the VR2332 strain. SC02-2025 was found to be a triple-recombinant virus （lineages 1.8/3/8.7）. This strain displayed the highest similarity to the NADC30 strain within the NSP2 and NSP3 regions， to the JXA1 strain within NSP1 and NSP7-10 regions， and the QYYZ strain in the remaining genomic segments. SC03-2025 showed 99% full-genome nucleotide similarity to the JXA1-P80 vaccine strain， with no evidence of recombination detected. This study， leveraging metatranscriptomic sequencing， precisely identified a mixed infection involving multiple PRRSV-2 lineages within a single pig farm in Sichuan. It revealed the complex epidemiological scenario involving the concurrent presence of a vaccine strain （JXA1-P80） and field strains （NADC30-like and QYYZ-like viruses）. These findings demonstrate that metatranscriptomic sequencing provides high-resolution diagnostic support for PRRSV disease surveillance and monitoring.

Porcine epidemic diarrhea virus （PEDV） is a coronavirus that causes acute diarrhea， vomiting， and dehydration in piglets， among other clinical signs， and has caused severe economic losses to the global swine farming industry. More and more studies have reported that long non-coding RNAs （lncRNAs） play important regulatory roles in porcine coronaviruses， however， the role and mechanism of lncRNA 663 in PEDV infection remain unclear. Based on prior transcriptome sequencing， we identified a co-expression pattern between lncRNA 663 and the tight junction protein CLDN10. This study aimed to explore their roles and regulatory relationship during PEDV infection. The qRT-PCR and Western blot results showed that the expression levels of lncRNA 663 and CLDN10 were up-regulated after PEDV infection of IPEC-J2 cells （P<0.05）， DSS treatment significantly reduced the expression levels of lncRNA 663 and CLDN10 （P<0.05）. Subcellular localization prediction revealed that lncRNA 663 was mainly localized in the cytoplasm， and RPISeq prediction revealed that lncRNA663 and CLDN10 were highly interoperable （SVM： 0.98， RF： 0.8）. Functional experiments demonstrated that siRNA-mediated knockdown of lncRNA 663 led to a significant decrease in both CLDN10 expression and PEDV replication （P<0.05）. Furthermore， structure prediction using AlphaFold3 and ChimeraX revealed potential hydrogen bond interactions between CLDN10 and the PEDV spike （S） protein， suggesting a role in viral entry or infection. Collectively， these results suggest that lncRNA 663 may promote PEDV replication by regulating CLDN10 expression. These findings expand our understanding of PEDV-host interactions and provide a theoretical basis for future antiviral strategies targeting host non-coding RNAs.

The aim of this study was to investigate the replication characteristics and pathogenicity of a cell-adapted strain derived from a prevalent PEDV isolate. The viral strain isolated from a large-scale pig farm was serially passaged in Vero cells to different generations. The biological characteristics of the virus were evaluated in vitro， and the 25th （F25） and 141st （F141） passages were inoculated into 3-day-old piglets to compare their pathogenic differences. The results showed that typical syncytial cytopathic effects （CPE） caused by PEDV were observed in Vero cells at the 4th blind passage of the clinical inoculum. By the 25th passage， both the CPE morphology and viral titers had stabilized. Upon reaching the 141st passage， the viral titer increased significantly and induced a more rapid cytopathic effect. Indirect immunofluorescence assay （IFA） specifically detected the expression of the PEDV N protein， and genomic sequence analysis identified the strain as genotype G2b. Pig challenge experiments demonstrated that， compared with the low-passage parental strain （YJH/2017/F25）， the YJH/2017/F141-inoculated group showed no significant clinical symptoms or histopathological lesions， along with a significant reduction in viral shedding via anal swabs. All assessed parameters showed no significant difference from those of the DMEM control group， indicating reduced pathogenicity and favorable safety profile of the strain. These findings indicate that after serial in vitro passaging， the prevalent PEDV strain acquired enhanced cell adaptability while exhibiting decreased pathogenicity in neonates. This study provides a promising candidate strain for the development of an attenuated PEDV vaccine， and its complete genomic sequence and biological characteristics establish a foundation for further research on recombinant viruses.

The non-structure protein （NSP） 12 of feline infectious peritonitis virus （FIPV） serves as the catalytic core component of the RNA-dependent RNA polymerase （RdRp）. However， the molecular mechanisms regulating its interactions with NSP7 and NSP8， as well as the enzymatic activity and function of the RdRp complex， remain to be fully elucidated. This study aims to investigate the molecular characteristics of NSP12， explore its interaction with NSP7 and NSP8， and verify the enzymatic activity of the RdRp complex. Firstly， the physicochemical properties and structural features of NSP12 were predicted by bioinformatics analysis. To investigate the interaction between NSP7， NSP8 and NSP12 in the FIPV RdRp complex， pcDNA3.1-Flag-NSP7， pcDNA3.1-HA-NSP8 and pcDNA3.1-Myc-NSP12 plasmids were constructed and expressed in 293T cells. The protein interactions and co-localization were detected using co-immunoprecipitation （Co-IP）， pull-down， and indirect immunofluorescence assay （IFA）. Secondly， the NSP7， NSP8 and NSP12 genes of FIPV were amplified by PCR and homologously recombined into the pET-30α vector to construct prokaryotic expression recombinant plasmids. The induced expression conditions were further optimized， and the proteins were purified by nickel column affinity chromatography and concentrated using an ultrafiltration tube to obtain the RdRp complex. Finally， an in vitro RNA elongation system for FIPV RdRp was established， and the enzymatic activity of the RdRp complexes obtained above was tested using the gel method. Bioinformatics analysis showed that NSP12 was a stable hydrophilic protein， containing 43.51% α helices， 45.02% random coils and 90 potential phosphorylation sites. The Co-IP， pull-down， and IFA assays confirmed the interaction and co-localization of NSP7， NSP8 and NSP12 in 293T cells. NSP7 and NSP8 were expressed as inclusion bodies， with molecular masses of 10 and 23 ku， respectively. In contrast， NSP12 was expressed as a soluble protein， with a molecular mass of 105 ku. The results of the enzyme activity assay demonstrated that the RdRp complex exhibited the capacity to extend the RNA template strand. Conversely， NSP12 alone exhibited no such activity. In this study， the FIPV RdRp complex was purified by prokaryotic expression and an enzyme activity detection system was established in vitro. It was confirmed for the first time that NSP12 from the FIPV-DF2 strain requires the formation of a complex with NSP7 and NSP8 to exert its RdRp function. This study lays the foundation for further exploring the biological function of FIPV RdRp and developing antiviral drugs targeting RdRp.

This study aimed to achieve soluble prokaryotic expression of the N-terminal extracellular domain of the pseudorabies virus （PRV） receptor Nectin1 protein， and to screen high-affinity， high-specificity DNA aptamers targeting it， thereby providing a novel molecular tool for blocking PRV adsorption. The porcine Nectin1 gene was cloned into the prokaryotic expression vector pET28a containing a maltose-binding protein （MBP） solubility-enhancing tag to construct the recombinant plasmid pET28a-MBP-Nectin1. The correctly digested and sequenced recombinant plasmid was transformed into Escherichia coli BL21 （DE3） for induced expression. The recombinant protein was validated by Western blot and pull-down assays. A DNA aptamer specifically targeting rMBP-Nectin1 was selected using magnetic bead-coupled Systematic Evolution of Ligands by EXponential enrichment （SELEX）. The affinity and specificity of the selected aptamer Apt-Nectin1-6 were evaluated by magnetic bead-based ELASA. Nectin1 knockdown cell lines （sh-Nectin1-1 and sh-Nectin1-2） were used as controls to verify the binding ability of Apt-Nectin1-6 to cell surface Nectin1 by flow cytometry. Additionally， the inhibitory effect of the aptamer on on adsorption of pseudorabies virus to host cells was assessed by flow cytometry and viral genome copy number analysis. Western blot and pull-down assays confirmed the correct expression and biological activity of the recombinant MBP-Nectin1 protein. Through SELEX， the DNA aptamer Apt-Nectin1-6， exhibiting high affinity to rMBP-Nectin1 with an equilibrium dissociation constant （Kd） of （0.11±0.02） nmol and no cross-reactivity with rMBP-gD， rMBP-S1， rMBP-CD163， or rMBP alone， was successfully screened. Molecular docking analysis indicated that Apt-Nectin1-6 binds to Nectin1 via hydrogen bonds. Further flow cytometry， viral genome copy number quantification， and virus titration collectively demonstrated that Apt-Nectin1-6 inhibits PRV adsorption to host cells in a dose-dependent manner. （P<0.05）. In this study， a DNA aptamer Apt-Nectin1-6 with high specificity and affinity for the Nectin1 protein was successfully screened and verified to effectively block PRV adsorption， providing a foundation for developing novel anti-PRV strategies.

This study aimed to analyze the genetic relatedness of key antigenic and virulence genes in the attenuated FJ19 strain of infectious laryngotracheitis virus （ILTV）， and to evaluate its safety and immunogenic efficacy in specific-pathogen-free （SPF） chickens， thereby providing valuable groundwork for the development of an attenuated live vaccine derived from the ILTV FJ19 strain. The ILTV FJ19 strain was adapted through allantoic cavity inoculation in chicken embryos， with sequencing confirming immunogenic and virulence genes. Safety tests were assessed in 14-day-old SPF chickens. Protective efficacy was demonstrated by challenging vaccinated chickens （ocular/oral routes at 14 days） with virulent ILTV strain at 21 days post-vaccination （dpv）. Vaccine durability was also determined. After serial passages in 10-day-old SPF chicken embryos via allantoic cavity inoculation， the EID₅₀ values of harvested P0， P1， P2， and P3 viral suspensions were 10^5.0， 10^6.5， 10^6.9， and 10^6.5·mL^-1， respectively. Genetic and phylogenetic analysis revealed： key immunogenic genes （gB， gC， gD， UL32） and virulence-associated genes （gE， gI， TK， ICP4， gG， gH， gK， gL， gM， gN） of FJ19 strain clustered closely with attenuated strains K317， Nobilis Laringovac^®， LT Blen， Poulvac ILT^®， Laryngo Vac and Chinese virulent isolates LJS09 （2009）， ck/CH/LHLJ/120305 （2012）， WF03 （2019）. Safety assessment demonstrated： Ocular inoculation （10^4.0 EID₅₀ per bird） caused mild conjunctivitis in 40% of 14-day SPF chickens， while oral administration showed no symptoms. Intratracheal challenge （10^4.0 EID₅₀ per bird） of FJ19 strain in 21-day-old SPF chickens caused transient respiratory reactions in a minority （30%）， with no severe ILTV-specific symptoms. Efficacy evaluation confirmed： Immunization （0.2 or 1 dose via ocular/oral routes） provided at least 90% protection against intratracheal challenge with virulent WF03 （10^4.0 EID₅₀ per bird） at 21 dpv， with no clinical signs in immunized groups. However， at least 90% of unimmunized virus-challenged chickens developed characteristic severe ILTV infection symptoms. Vaccine-induced immunity persisted for ≥6 months. The results demonstrated that： The ILTV strain FJ19 achieves high-titer cultivation via allantoic cavity inoculation. Its major antigenic and virulence genes show no significant variation compared to early Chinese isolates and vaccine strains. The FJ19 strain demonstrated excellent safety and immunogenicity in 14-day-old SPF chickens， immunity duration reached 6 months， with oral administration maintaining safety and efficacy. Thus， adaptation to allantoic cavity inoculation of FJ19 strain yielded a virus suspension with optimal safety and immunogenicity， the attenuated ILTV FJ19 strain represents an ideal candidate for oral attenuated live vaccine development.

Infectious bursal disease （IBD） severely restricts the economic development of the poultry industry. Therefore， the development of safe and efficient IBD vaccines has become a key topic of considerable interest. To explore the feasibility of using Eimeria tenella as a live vector for the expression and delivery of infectious bursal disease virus （IBDV） antigens， this study constructed a genetically modified strain of E. tenella （Et-VP2） expressing the VP2 protein of the IBDV. An expression plasmid was constructed， digested with enzymes， and then transfected into E. tenella using nuclear transfection. Flow cytometry was utilized to sort and select positive strains expressing the red fluorescent reporter gene （mCherry）， and the expression of the VP2 gene was identified at both the genetic and protein levels. Results showed that after three passages in vivo in chicks， the transgenic E. tenella expressing VP2 protein had a positive rate of fluorescence exceeding 95%， confirming successful integration and stable expression of the foreign gene at both the genetic and protein levels （with fluorescence rates reaching 95% in the offspring population）. Indirect immunofluorescence results indicated that the fusion protein （Mic2-linker-VP2） was localized at the apical end of the sporozoites of E. tenella. This study successfully established a stable transfection system for E. tenella， laying the foundation for subsequent research on the immune protection of genetically modified strains against infectious bursal disease.

Rabbit coccidiosis is a worldwide-distributed parasitic protozoal disease， which has caused huge economic losses to rabbit industry. Traditional methods of anticoccidial treatment result in drug resistance and drug residue， vaccination is a promising alternative. The rEiAMA1， rEiIMP1， EmGAM56， and EmROP17 were respectively displayed on the surface of B. subtilis spores by using a coat protein （CotB） as an anchor. Then the recombinant spores were used to oral immunize rabbits， the protective effects of B. subtilis-rEiAMA1， -rEiIMP1， and -rEiAMA1+rEiIMP1 against Eimeria intestinalis were evaluated； and the protective effects of B. subtilis-EmGAM56， -EmROP17， and -EmGAM56+ EmROP17 against Eimeria magna were evaluated. The results showed that B. subtilis-rEiAMA1 immunized rabbits displayed better anti-E. intestinalis protective effect， the oocyst output and lesion score were significantly decreased （P<0.05）， the oocyst output decrease ratio was 59.91%. B. subtilis-EmGAM56 immunized rabbits displayed better anti-E. magna protective effect， the oocyst output was significantly decreased （P<0.05）， the oocyst output decrease ratio was 68.54%. Moreover， the immunized rabbits showed significantly increased specific serum IgG levels and specific IgA levels （P<0.05）. The four recombinant B. subtilis spores can stimulate the body to produce specific intestinal and systemic humoral immune responses. B. subtilis-rEiAMA1 and B. subtilis-EmGAM56 showed better immunoprotection against E. intestinalis and E. magna， respectively. Therefore， B. subtilis is a potential live vector for rabbit coccidiosis vaccines.

This study aims to evaluate the biological characteristics of Trichinella spiralis nuclear hormone receptor-23 （NHR-23） and its potential for vaccine development. The recombinant TsNHR-23 protein （rTsNHR-23） was obtained via cloning and expression， followed by purification. Its immunoreactivity was assessed via Western blot， while transcriptional levels of the TsNHR-23 gene across different developmental stages of T. spiralis were analyzed using qPCR. In vitro experiments were conducted to analyze the inhibitory effect of anti-rTsNHR-23 polyclonal antibody on the molting of muscle larvae and to explore the effect of this protein on the function of mouse macrophages. The immune-protective efficacy of PLGA nanoparticle-encapsulated rTsNHR-23 was tested by immunizing ICR mice and analyzing parasite burden. The TsNHR-23 gene was transcribed across all developmental stages of T. spiralis， with the highest expression observed in muscle larvae （ML） stage. Anti-rTsNHR-23 antibodies significantly reduced larval molting rates and inhibited molting progression. The rTsNHR-23 protein significantly enhanced the transcription levels of IL-1β， IL-6， and IL-10 in mouse macrophages. Immune protection experiments demonstrated that mice immunized with the rTs-NHR-23 protein exhibited a highly significant increase in serum IgG antibody levels compared to the control group. After the first immunization， IGF-1 levels markedly decreased， while IL-6 and TGF-β levels increased. Following the second immunization， IGF-1 partially rebounded， whereas TGF-β showed significant downregulation. Post-challenge infection， compared to the control group， the immunized mice displayed significant reductions in the number of T.spiralis adult worms， the average number of newborn larvae produced per female worm， and the muscle larval burden. rTsNHR-23 exhibits potent inhibitory effects on ML molting， modulates macrophage functions， and significantly reduces parasitic load in immunized hosts， demonstrating promising potential as a candidate vaccine against T. spiralis.

This study aimed to identify the suspected pathogens causing rabbit mortality in a large-scale meat rabbit farm in Ningxia and analyze their pathogenicity. The causative pathogens and their pathogenicity were determined through necropsy of deceased rabbits， bacterial isolation and culture， 16S rRNA sequencing analysis， PCR identification， antimicrobial susceptibility testing， and challenge experiments in mice and rabbits. Results showed that 3 Gram-negative bacterial strains were isolated and identified as capsular type A Pasteurella multocida， Proteus mirabilis， and Escherichia coli through 16S rRNA sequencing and PCR identification. These strains were designated as QF24-Pm01， QF24-Ps02， and QF24-Ec03， respectively. Antimicrobial susceptibility testing by the Kirby-Bauer （K-B） method revealed that QF24-Pm01， QF24-Ps02， and QF24-Ec03 exhibited resistance to commonly used clinical drugs such as penicillin， kanamycin， and enrofloxacin， but were sensitive to ceftriaxone. Detection of resistance genes showed that all three isolates carried resistance genes aadB， strA， strB， bla_TEM， and sul1. Virulence gene analysis indicated that Pasteurella multocida carried 16 virulence genes， including exbB， exbD， and hgbA； Proteus mirabilis carried ureC， zapA， atfC， atfA， and pmfA； and Escherichia coli carried eaeA and fimC. Mouse pathogenicity experiments revealed LD₅₀ values of 9.27×10⁵ CFU， 1.43×10⁶ CFU， and 1.88×10⁷ CFU for QF24-Pm01， QF24-Ps02， and QF24-Ec03， respectively. The results of the mixed-infection pathogenicity assay in mice showed that mortality occurred at 9 h， 18 h， and 16 h post-inoculation in the QF24-Pm01， QF24-Ps02， and QF24-Ec03 groups， respectively. In contrast， the QF24-Pm01+QF24-Ec03 and QF24-Ps02+QF24-Ec03 co-infection groups exhibited mortality at 6 h and 13 h post-inoculation， respectively. Histopathological analysis revealed that both single and mixed infections resulted in tissue damage in the lungs， liver， and intestines. The primary pathological changes included pulmonary interstitial edema， capillary congestion， and neutrophil infiltration； hepatic steatosis and disordered hepatocyte cord arrangement； as well as intestinal epithelial shedding and villous rupture. In the rabbit pathogenicity assay， all rabbits in the QF24-Pm01 + QF24-Ec03 co-infection group and the QF24-Pm01 group succumbed at 24 and 36 h post-inoculation， respectively. The mortality rates at 7 days post-inoculation for the QF24-Ps02+QF24-Ec03， QF24-Ec03， and QF24-Ps02 groups were 60%， 20%， and 40%， respectively. Necropsy findings demonstrated that rabbits in the QF24-Pm01+QF24-Ps02+QF24-Ec03 co-infection group exhibited pulmonary congestion and edema with a ground-glass appearance， as well as splenic atrophy and dark brown discoloration. These pathological changes were consistent with the lesions observed in clinical necropsy examinations of affected rabbits. This study demonstrates that the pathogenicity of Pasteurella multocida is enhanced when co-infected with Escherichia coli， and that Escherichia coli and Proteus mirabilis exhibit increased virulence when co-infected. Therefore， co-infection with Pasteurella multocida，Proteus mirabilis， and Escherichia coli may be the primary cause of high mortality rates in meat rabbits at the studied facility. Consequently， in addition to preventing Pasteurella multocida infection， control measures against Escherichia coli and Proteus mirabilis should be reinforced to reduce the risk of mixed infections and associated mortality.