Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is one of the major pathogens threatening the global swine industry and causes enormous economic losses annually. Vaccination remains one of the most important strategies for the prevention and control of PRRSV infection. In this study, a novel genotype 2 NADC30-like PRRSV strain, designated AHFY, was isolated and characterized from eastern China. Phylogenetic analysis revealed that strain AHFY belonged to sublineage 1.8. An attenuated strain, AHFY-F140, was obtained through 140 serial passages in MARC-145 cells. Genomic sequence comparison between AHFY-F140 and the parental strain AHFY-F1 identified two discontinuous amino acid (aa) deletions in the nonstructural protein 2 (NSP2) gene, spanning residues 355-364 and 391-583, which were first detected at passage 90 and persisted through passage 140. The viral titer gradually increased with passaging, rising from 104.3 TCID50/mL for the parental strain AHFY-F1 to 108.7 TCID50/mL for AHFY-F140. A pathogenicity evaluation was conducted in 4-week-old PRRSV-free New American Line piglets using AHFY-F1 and AHFY-F140. Piglets inoculated with AHFY-F1 developed persistent fever and obvious respiratory clinical signs, accompanied by severe and typical histopathological lung lesions. In contrast, piglets inoculated with AHFY-F140 showed no fever or other clinical signs. PRRSV-specific antibodies became positive at 11 days postinoculation (dpi) in AHFY-F140-inoculated piglets, which was later than the 7 dpi observed in AHFY-F1-inoculated piglets. Furthermore, immunization with AHFY-F140 provided robust protection against subsequent challenge with either the parental strain AHFY-F1 or the highly pathogenic PRRSV (HP-PRRSV) strain JXA1 (lineage 8). Collectively, these findings demonstrate that the attenuated strain AHFY-F140 represents a promising vaccine candidate against both NADC30-like and JXA1-like PRRSV infections.

Classical Swine Fever (CSF), or swine cholera, is a highly acute, febrile, and contagious disease caused by the Classical Swine Fever virus (CSFV) in pigs. A total of 205,622 samples were collected from 689 pig farms defined as individual production units nationwide across 20 Chinese provinces/cities from January to December 2022. The results showed that at the sample level, the prevalence of CSFV was 0.25

The African swine fever virus (ASFV) employs sophisticated strategies to promote viral replication in the host; however, the underlying mechanisms remain incompletely understood. Here, we demonstrated that the ASFV encoded pE199L protein acts as a potential mitophagy receptor that disrupted innate immunity through structural mimicry. The pE199L protein localized to mitochondria via its C terminal hydrophobic domain (155-199 aa) and induced mitochondrial fission by promoting DNM1L/Drp1 phosphorylation. Importantly, pE199L contained three LC3-interacting regions (LIRs: W35-I38, F157-L160, F193-L196) that directed autophagic degradation of key immune adaptors. Specifically, pE199L mediated mitophagic clearance of TBK1 (the CGAS-STING1 pathway) and MAVS (the RLR-MAVS pathway), thereby inhibiting type I interferon production and enhancing viral replication. This dual degradation mechanism was confirmed through rescue experiments using autophagy inhibitors and functional assays with LIR mutants. We identifted pE199L as the first canonical mitophagy receptor encoded by ASFV, unveiling a novel immune evasion strategy and a potential target for antiviral vaccine development.Abbreviations: 3-MA: 3-methyladenine; aa: amino acid; ASFV: African swine fever virus; CGAS: cyclic GMP-AMP synthase; co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DNM1L/Drp1: dynamin 1 like; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; hpi: hour post-infection; IFN: interferon; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; Mdivi-1: mitochondrial division inhibitor 1; MOI: multiplicity of infection; MT-CO2/COXII: mitochondrially encoded cytochrome c oxidase II; PINK1: PTEN induced kinase 1; poly(dA:dT): poly(deoxyadenylic-thymidylic) acid; poly(I:C): polyinosinic-polycytidylic acid; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TOMM20: translocase of outer mitochondrial membrane 20; WT: wild-type.

Senecavirus A (SVA) causes considerable economic losses in the swine industry, particularly among piglets. However, effective serological diagnostic assays for SVA are still lacking. A testing platform based on nanobody-horseradish peroxidase (Nb-HRP) fusion protein-based competitive ELISA (cELISA) for the detection of antibodies against various animal viruses is available. In this study, a rapid and reliable cELISA was established using this platform for specific detection of antibodies against SVA. Two anti-SVA nanobodies were selected from an immunized Bactrian camel using phage display technology. The Nb84-HRP fusion protein was expressed and used as a probe during the process. The assay exhibited a cut-off value of 19.4

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major threat to global swine production. In this study, a novel strain (PRRSV-AH1) was isolated during a respiratory disease outbreak at a commercial swine operation in Anhui Province, China. Viral replication in MARC-145 cells was confirmed by observing cytopathic effects (CPEs) and conducting immunofluorescence assays (IFAs). Whole-genome sequencing revealed a 15,020 bp genome exhibiting 90.0% identity with the NADC30 reference strain, including lineage 1-characteristic nonstructural polyprotein (Nsp)2 deletions. A distinctive L10S substitution in GP2 aligned with conserved residues of PRRSV-1. Recombination analysis identified PRRSV-AH1 as a novel chimera with a NADC30-like backbone incorporating CH-1a-like (lineage 8), JXA1-like (lineage 8), and QYYZ-like (lineage 3) sequences-representing the first reported instance of this specific recombination pattern. Experimental infection of piglets induced characteristic PRRSV pathology, including sustained pyrexia, reduced weight gain, prolonged viremia, and neutralizing antibody seroconversion. Comparative pathogenicity analysis revealed that the PRRSV-AH1 strain elicited febrile responses and peak body temperatures intermediate between classic NADC30-like strains and JXA1 strains. Notably, PRRSV-AH1 demonstrated a PRRSV-N-specific IgG induction capacity comparable to that of highly pathogenic variants. These findings establish PRRSV-AH1 as a multilineage recombinant (NADC30-like, CH-1a, QYYZ, and JXA1 Lineages) resulting from multiple genetic exchanges, underscoring the increasing complexity of PRRSV diversity in China. Accelerated mutation and recombination across lineages complicate disease control efforts, emphasizing the need for enhanced surveillance, mechanistic recombination studies, and the development of novel vaccines to mitigate future outbreaks.

African swine fever virus (ASFV), a highly contagious double-stranded DNA virus, causes a hemorrhagic disease with mortality approaching 100% in domestic pigs. Despite intensive efforts, effective vaccine options remain limited, highlighting the need for continuously identifying viral neutralizing epitopes. Nanobodies can effectively engage cryptic epitopes that are often inaccessible to conventional antibodies due to their small size. Here, we immunized a Bactrian camel with recombinant ASFV H240R expressed in a bacterial system, and an H240R-specific VHH phage-display library was constructed and screened, yielding a nanobody named H240R-Nb82. H240R-Nb82 efficiently neutralized distinct ASFV isolates (genotypes Ⅰ, Ⅱ and their recombinants) in porcine alveolar macrophages (PAMs). Mechanistically, H240R-Nb82 inhibited the early stage of ASFV infection by blocking viral attachment to host cells. Epitope mapping using truncation analysis and site-directed alanine mutagenesis showed that H240R-Nb82 binds to a linear, native virion-accessible conserved region within H240R (residues 33GHYPFSFELK42) and His34 made an important contribution to H240R-Nb82 binding. H240R-Nb82 also directly bound purified ASFV virions. Moreover, immunization of mice with the epitope-containing antigen induced sera capable of neutralizing ASFV infection in PAMs, demonstrating the protective potential of this conserved determinant. Collectively, these findings define a previously unrecognized conserved neutralizing epitope on ASFV H240R and provide a new target protein for developing ASFV subunit vaccines.

IntroductionPorcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen threatening the global swine industry, with existing vaccines failing to provide broad protection due to the high viral genetic variability and immune evasion capacity. Retinoic acid (RA), a vitamin A metabolite known for immunomodulatory functions, has been reported to enhance antiviral innate immunity and suppress excessive inflammation. However, whether RA affects PRRSV infection remains unclear.MethodsThe antiviral efficacy of all-trans retinoic acid (ATRA), the major biologically active form of RA, against PRRSV infection was evaluated in both MARC-145 cells and primary porcine alveolar macrophages (PAMs). Furthermore, transcriptomic profiling was conducted to investigate the regulatory effects of ATRA on uninfected and PRRSV-infected PAMs.ResultsATRA significantly inhibited PRRSV infection and replication with minimal cytotoxicity. Transcriptomic profiling revealed that ATRA upregulated basal immune signaling and metabolic transport pathways while suppressing non-specific inflammatory activity in uninfected PAMs. Interestingly, comparative transcriptomics revealed that PRRSV infection led to hyperinflammatory responses and disrupted lipid homeostasis in PAMs, while ATRA treatment reversed these alterations.DiscussionOur findings elucidate that RA exerts antiviral effects against PRRSV through a dual mechanism of attenuating excessive inflammation and restoring cellular metabolic homeostasis, highlighting its potential as an immunomodulatory agent for viral infection control.

Fowl adenoviruses (FAdVs) are widely distributed in poultry populations around the world, and many diseases are associated with FAdV infection in chickens. This study documented the first characterization of coinfection with fowl adenovirus serotypes 1 and 4 (FAdV-1 and -4) associated with hydropericardium hepatitis syndrome (HHS) in Chinese layer flocks, revealing a novel viral cooperation mechanism. Two novel strains (CH/SX/201805-1 and -4) were identified and isolated, with whole-genome sequencing showing CH/SX/201805-1 clustering with FAdV-1 (99.7% identity to FAdV-A-61/11z), whereas CH/SX/201805-4 displayed characteristic ORF19/27/29 deletions mirroring emergent Chinese FAdV-4 variants. Experimental coinfection in SPF chickens resulted in 87.5% mortality, which was 16.7% greater than that resulting from infection alone, with exacerbated pathology. In vitro coinfection experiments demonstrated concurrent viral replication within same LMH cells, a previously unreported phenomenon, where FAdV-1 increased FAdV-4 replication efficiency 21-fold (p < 0.001). Transcriptomic profiling revealed heat shock protein A2 (HSPA2) as the most differentially expressed gene, which was upregulated 2.8-fold during coinfection compared with infection with FAdV-4 alone. Functional validation through HSPA2 knockdown reduced FAdV-4 replication, establishing that FAdV-1 potentiates FAdV-4 through HSPA2-mediated host modulation. These findings provide the first evidence of HSPA2-dependent interserotype synergy in FAdV and can be used to develop a cellular model for FAdV coinfection studies. These insights redefine the understanding of FAdV pathogenesis and create new avenues for targeted intervention strategies against emerging FAdV coinfections.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is threatening public health globally. Due to the shortage of efficient in vitro cell culture systems and in vivo model, the viral replication and pathogenesis mechanisms remain largely unknown. Here, we found that HEV-ORF3 protein showed prion-like properties in HEV-infected cells and existed as both monomer and SDS-resistant aggregate forms. In an in vitro cell-free model, incubation of ORF3 monomer with its aggregates could effectively convert ORF3 monomer into aggregates, mirroring a typical characteristic of prion. In addition, the prion domain (PrD) of a classic yeast prion Sup35 could be functionally replaced by full-length HEV-ORF3 or its N-terminal candidate PrD (cPrD). An F10S substitution in the ORF3-cPrD impaired HEV-ORF3 aggregation propensity and blocked the function of ORF3 in enhancing the stability of microtubules in HEV-infected cells, thus led to the inhibition of viral capsid translocation to microtubules and virion release from infected cells. In Mongolian gerbil models, HEV bearing ORF3F10S mutation demonstrated attenuated virulence in vivo compared with wild-type HEV, as evidenced by reduced viremia and viral shedding, as well as alleviated pathological changes of liver tissue in gerbils infected by HEV-ORF3F10S mutant. In conclusion, our data suggest that HEV-ORF3 is a prion-like protein which is involved in viral capsid translocation and virion release, supporting the hypothesis that the self-propagating properties of prion proteins or prion-like proteins are widely exploited in nature and play diverse roles in physiological function.

In 2020, severe diarrhea occurred in four-month-old fattening pigs from nine farms in Shandong Province, China. Fecal samples were collected from diseased pigs and tested by PCR for the presence of mammalian orthoreovirus (MRV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine rotavirus A (PoRVA), transmissible gastroenteritis virus (TGEV), porcine kobuvirus (PKV), and pseudorabies virus (PRV). The viral RNA of MRV and PEDV was detected in the fecal samples. The genome sequences of MRV and PEDV were successfully amplified from the same fecal sample. Genomic and phylogenetic analysis showed that the MRV isolate named MRV2-SD/2020 belongs to serotype 2 MRV (MRV2) and may originate from the reassortment of human and porcine MRVs. Compared with other MRV2 strains, there were four other unique amino acid mutations (L274I, F302L, V347I, and T440M) in the receptor binding region. For the PEDV isolate named PEDV-SD/2020, the nearly complete genome was amplified from the positive fecal samples. Phylogenetic analysis showed that it was classified into the G2a genotype. Compared with CV777 and other PEDV variant strains, its spike (S) protein exhibited two unique mutations (S663T and L966M). This study first reports the co-infection of PEDV and MRV2 in the pigs and provides a new direction for the prevention and control of the diarrhea diseases.

Currently, there are no commercial serologic marker or differentiation of infected and vaccinated animal (DIVA) vaccines for the eradication of porcine reproductive and respiratory syndrome virus (PRRSV) infection from pig farms. In a previous study, a nanobody-based competitive ELISA (cELISA) was specifically developed to detect anti-genotype 2 PRRSV (PRRSV-2) antibodies. On the basis of the epitope recognized by the nanobody and the prevalence of PRRSV-2 infection in China, a DIVA vaccine candidate strain was designed and evaluated in the present study. First, an infectious cDNA clone based on the genomic sequence of the highly pathogenic PRRSV-2 (HP-PRRSV) isolate SX-HD was constructed and named rSX-HD. Using the infectious clone as the backbone, a chimeric infectious cDNA clone in which the gene encoding the nucleocapsid (N) protein was replaced with the gene encoding the genotype 1 PRRSV N protein was generated and named rSX-HD2M1. The chimeric PRRSV rSX-HD2M1 was subsequently rescued successfully in Marc-145 cells, which were then passaged for 120 generations for attenuation. A safety study indicated that rSX-HD2M1-F120 is not pathogenic to piglets. In vivo inoculation and challenge experiments suggested that rSX-HD2M1-F120 vaccination significantly reduced serum viral loads and lung tissue lesions and that vaccinated piglets did not show any clinical symptoms or histopathological changes. Furthermore, this recombinant marker virus, in conjunction with the previously developed nanobody-based cELISA, enables serological differentiation between marker virus-infected animals and those infected with wild-type PRRSV-2. These results suggest that rSX-HD2M1-F120 is a good candidate for providing a live attenuated DIVA vaccine against PRRSV-2 infection in piglets.

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. PCV3 capsid protein (Cap) can self-assemble into viruslike particles (VLPs), and is an ideal candidate for vaccines and diagnostic reagents.In this study, the recombinant PCV3 Cap protein was successfully expressed in E. coli by deleting the nuclear localization sequence (NLS). The PCV3 VLPs were observed by transmission electron microscopy, and its immunogenicity was evaluated in sixweek-old female BALB/c mice. A monoclonal antibody was named mAb 2D6, and demonstrated strong reactivity and specificity to PCV3 Cap. The purified mAb 2D6 was further used for bio-panning to select phage expressing specific epitopes from phage-displayed 7 mer-peptide library. A novel linear B-cell epitope, recognized by mAb 2D6, was identified at the amino acid region 47-53 of Cap. The phage peptide sequences were analyzed using multiple sequence alignment and evaluated by peptide ELISA. These results provide insights for developing diagnostic tools and potential vaccines for PCV3.

ABSTRACT In poultry, fowl adenovirus (FAdV) and co-infected viruses (such as avian hepatitis E virus, aHEV) are likely to cause decreased egg production, inclusion body hepatitis, and pericardial effusion syndrome. From July to September 2023, eight poultry farms of commercial broilers and commercial layers suffered from increased mortality, decreased egg production, and the presence of hydropericardium-hepatitis syndrome-like gross lesions in Shaanxi province, China. To determine the source of the infection, the viruses of aHEV, FAdV, avian leukosis virus (ALV), Marek’s disease virus (MDV), Newcastle disease virus (NDV), and H9N2 avian influenza virus (AIV) were detected. A total of 240 liver samples were collected from eight farms and detected by molecular etiology methods. The samples were tested positive for FAdV and aHEV and negative for NDV, MDV, ALV, and H9N2 AIV. Phylogenetic analysis based on the hexon loop-1 gene showed that these isolates were clustered into three distinct serotypes: FAdV serotype 4 (FAdV-4), FAdV-8a, and FAdV-8b. The partial ORF2 gene sequences obtained in this study shared the highest identity (76%–97%) with corresponding sequences of other known avian HEV isolates and belonged to gene type 3 avian HEV. Meanwhile, the isolated FAdV in this study could multiply in leghorn male hepatoma (LMH) cells. Overall, the disease may be mainly caused by FAdV and aHEV coinfection on these farms, and this is the first study to discover FAdV-4/8a/8b and avian HEV coinfection in a farm in China. IMPORTANCE Last year, eight poultry farms of commercial broilers and commercial layers suffered a serious disease in Shaanxi province, China. After molecular etiological investigation, the disease may be mainly caused by fowl adenovirus (FAdV) (FAdV-4, FAdV-8a, and FAdV-8b) and avian hepatitis E virus (aHEV) (genotype 3 avian HEV) coinfection on these flocks, and this is the first study to find FAdV-4/8a/8b and avian HEV coinfection in a farm. It provides a valuable foundation for the prevention and control of FAdV and avian HEV coinfection in chicken farms.

Hepatitis-hydropericardium syndrome, caused by fowl adenovirus serotype 4 (FAdV-4), has resulted in significant economic damage to the poultry industry. To monitor viral exposure and vaccine efficacy, some traditional antibody-based immunoassays have been developed for detecting anti-FAdV-4 antibodies. However, these assays have some drawbacks including multi-step operations and higher production cost. Recently, nanobodies are regarded as a promising tool for developing immunoassays. In the study, 23 nanobodies against FAdV-4 were screened and expressed with horseradish peroxidase (HRP) in the HEK293T cells. Then, the FAdV-4-Nb28-HRP fusion protein was selected for developing competitive enzyme-linked immunoassays (cELISA) to detect anti-FAdV-4 antibodies in the chicken sera. The optimal concentrations and dilutions for the coating antigen, fusion protein and testing sera were determined to be 400 ng/well, 1:80 and 1:20, respectively. After the coated plates were vacuumized and stored, the operation of cELISA to detect clinical chicken sera was only one-step and the full time was 75 min. The cELISA also exhibited high sensitivity, specificity, reproducibility and good agreement with the commercial ELISA kit. When the sequential sera from the challenged chickens were tested, the cELISA showed superior sensitivity compared with the commercial ELISA kit. Moreover, epitope mapping revealed that the nanobody specifically recognized the sites GLN235 ASN236 SER238 of the fiber-1 protein, highly conserved among different FAdV-4 isolates and different from the FAdV-1 and -8. The results indicated that cELISA can specifically detect anti-FAdV-4 antibodies. Collectively, the developed one-step nanobody-based cELISA is an ideal method for epidemiological investigation and vaccine immune evaluation of FAdV-4.

BACKGROUND:Hepatitis E virus (HEV) is a zoonotic pathogen, and its infection is widespread in China. However, few studies were carried out on pets, especially in western China. Therefore, this study aimed to characterize the prevalence of HEV infection in pet dogs, cats, and rabbits. Serum samples from 177 pet dogs, 98 pet cats, and 56 pet rabbits were collected from a pet hospital located in Xi'an city of Shaanxi province, western China, to detect anti-HEV antibodies and HEV RNA. RESULTS:The positivity rates of HEV IgG were 18.08% (32/177), 16.33% (16/98), and 10.71% (6/56) in pet dogs, cats, and rabbits, respectively. In addition, higher OD values of the anti-HEV IgM antibody were chosen for HEV detection, and the HEV RNA positivity rates were 0% (0/177), 0% (0/98), and 8.93% (5/56). Meanwhile, two different partial HEV ORF1 genes were obtained from 5 positive pet rabbit samples. These sequences shared 88.2% identity with each other and 48.6-98.2% identity with other HEVs. Phylogenetic analysis revealed that CN-SX-R1 and CN-SX-R2 (GenBank Nos. PP982770 and PP982771) detected in this study were closely related to genotype 3 HEV strains and belonged to rabbit HEVs. CONCLUSIONS:These results showed that HEV was prevalent in pet dogs, cats, and rabbits in Shaanxi, suggesting that necessary measures must be taken to prevent and control HEV infection in these species.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes abortion and respiratory disease in swine, hindering the development of the pig farming industry worldwide. However, at present, there are no effective vaccines or drugs for PRRSV control. In this study, we evaluated the inhibitory effect of hyperoside on PRRSV replication in vitro and in vivo and explored the underlying mechanisms. Our results revealed that hyperoside significantly inhibited PRRSV infection in MARC-145 and porcine alveolar macrophages (PAMs). This inhibition was linked to the hyperoside-induced attenuation of pro-inflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) upregulation induced by PRRSV infection, which was mediated by the suppression of the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-kB) signaling pathway. Moreover, hyperoside alleviated the autophagy induced by PRRSV via p62/Nrf2/Keap1 signaling pathway activation. In vivo, hyperoside treatment led to an obvious decrease in PRRSV replication in piglets. Therefore, hyperoside may be a useful antiviral agent against PRRSV.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) causes abortion and respiratory disease in swine, which induces huge economic losses every year. However, there have been no effective vaccines or drugs for PRRSV control until now. Our present study found that the inhibitory effect of hyperoside on PRRSV replication in vitro and in vivo. Furthermore, we demonstrate that hyperoside inhibits PRRSV proliferation via inhibiting inflammation and autophagy through the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) and p62-Nrf2-Keap1 signaling pathways. Hence, we believe that hyperoside may be a useful antiviral agent to control PRRSV.

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), can induce 80–100% mortality in newborn piglets; therefore, specific and rapid detection methods are important for the prevention of this viral infection. In particular, methods for detecting neutralizing antibodies (nAbs) can be used to evaluate the immunization effect of PEDV vaccines. The spike protein of PEDV (PEDV-S) has been universally used as an antigen to develop immunoassays to detect nAbs. Nanobodies (Nbs) offer advantages such as ease of genetic engineering and low production costs, making them promising for diagnostic applications. In this study, PEDV-S was expressed via the baculovirus system and was used as an antigen to immunize Bactrian camels. A total of 10 Nbs against PEDV-S were first screened and expressed as fusion proteins with horseradish peroxidase (HRP) in HEK293T cells. A Nb-HRP fusion protein named PEDV-S-Nb13-HRP was subsequently selected and used as a probe for developing a competitive enzyme-linked immunosorbent assay (cELISA) to detect anti-PEDV nAbs. Optimization assays identified 80 ng/well of PEDV-S as the optimal coating antigen concentration. The optimal dilution of PEDV-S-Nb13-HRP was 1:200, and the optimal serum dilution was 1:10. The cutoff value of cELISA was determined as 28.1%, demonstrating high specificity, repeatability, stability, and good agreement rates with two commercial ELISA kits (93.6%) and a serum neutralization test (96.34%). Additionally, the results of the detection of IgA antibodies in oral and milk samples from sows were in good agreement with those of the IDEXX PEDV IgA kit. These results demonstrate that the cELISA is a reliable and cost-effective method for detecting anti-PEDV nAbs.

Immunoassay is a diagnostic tool based on the specific binding of antibodies and antigens with widespread applications. Nonetheless, several research obstacles, like poor specific antibodies, the poisonous reagents and unstable results, still remain challenges. Herein, we innovatively reported a colorimetric and fluorescent dual-mode immunoassay based on the bifunctional nanobody for SEA detection. Benefiting from the advantages of nanobodies, the bifunctional protein with both recognition and catalysis was built to identify and catalyze with efficiency to generate the first colorimetric signal. Meanwhile, the introduction of quinine as the natural source of the second fluorescent signal greatly improved the stability and safety of detection. In addition, the proposed method was successfully applied to detecting SEA in food samples with high accuracy and stability. This study integrated the bifunctional nanobody with eco-friendly fluorescent product to provide a specific and green platform for the detection of foodborne toxins.

The wild-type H1N1 and H3N2 swine influenza virus (SIV) strains are unsuitable for vaccine production because of high lethality in chicken embryos and low reproductive titers. This study developed recombinant H1N1-Re1 and H3N2-Re1 strains via HA and NA genes from the wild-type H1N1 SW/GX/755/17 and H3N2 SW/GX/1659/17 strains combined with six internal genes from the H1N1 A/PR/8/34 strain. The recombinant viruses demonstrated typical cytopathic effects in MDCK cells, and the presence of viral particles was confirmed via electron microscopy. Growth curve analysis revealed titers of 108.31 and 108.17 EID50 per 100 µL for H1N1-Re1 and H3N2-Re1, respectively, within 72–96 h postinoculation. Virus stocks were used to produce a bivalent inactivated vaccine. After two immunizations, hemagglutination inhibition titers in piglets were significantly greater than those induced by commercial vaccines and were sustained from 5 to 29 weeks postimmunization. Upon challenge with virulent wild-type SIV strains, viral isolation occurred in all pigs in the PBS group (5/5 protection), whereas no virus was detected in the bivalent vaccine group (0/5). In contrast, the commercial vaccine group had a viral isolation rate of 1/5. Pathological examination revealed severe pulmonary lesions in the PBS group, mild changes in the commercial vaccine group (1/5), and normal lung morphology in the bivalent vaccine group. This study demonstrated the successful application of an eight-plasmid reverse genetics system to develop recombinant vaccine strains with enhanced immunogenicity and replication efficiency. The bivalent inactivated vaccine provides prolonged and complete protection against H1N1 and H3N2 SIV strains, offering a robust tool for controlling evolving SIV variants.

Porcine reproductive and respiratory syndrome virus (PRRSV) imposes substantial economic losses on global swine production. While modified live vaccines remain the primary prevention tool, their efficacy is compromised by the genetic variability of PRRSV. This study developed a broadly neutralizing monoclonal antibody (mAb) that targets a conserved viral epitope as an alternative therapeutic strategy. Using hybridoma technology, BALB/c mice with purified PRRSV particles and screened mAb-5F2 is immunized, which demonstrated cross-lineage neutralization against PRRSV-2 (lineages 1, 5, and 8) and PRRSV-1 strains. Epitope mapping revealed two residues, Q48 and I50, of GP4, which are key motifs for interaction with mAb-5F2. Mechanistic studies revealed that the mAb-5F2 sterically hinders the GP4-CD163 interaction, reducing viral entry efficiency by 78-85% at a concentration of 50 µg mL-1. To enhance clinical applicability, a porcine antibody (5F2-pFc) is engineered. In vivo challenge trials revealed that 5F2-pFc treatment completely prevented mortality in PRRSV-infected piglets (100% survival vs 40% in controls) while significantly reducing pulmonary viral loads (2-log decrease) and histopathological lesions. This work identified a novel conserved neutralizing epitope on PRRSV GP4 and established a porcineized antibody platform that combines broad-spectrum neutralization with clinical practicality, offering a promising strategy to overcome current vaccine limitations in PRRSV management.