Pseudorabies virus (PRV) is one of the most significant pathogens threatening the swine industry, and its infection has caused substantial economic losses to pig farming worldwide. This study utilized two PRV strains isolated by our laboratory: the GXLB-2015 strain, a natural recombinant between a PRV variant strain and the Bartha-K61 vaccine strain, which exhibits stronger virulence, and the GXGG-2016 strain, a classical genotype II PRV strain with a natural 69-amino acid deletion in its TK gene, which demonstrates weak virulence and is non-pathogenic in mice. Focusing on endoplasmic reticulum stress (ERS) and cellular autophagy, this research explored the molecular mechanisms underlying the significant difference in virulence between these two PRV strains. The results revealed that infection with both strains induced noticeable ERS and autophagy, both of which inhibited viral replication. Further investigation showed that upon ERS induction, the GXLB-2015 strain mediated autophagy by activating both the PERK-eIF2α-ATF4-CHOP and IRE1-XBP1 pathways; inhibiting these pathways suppressed viral replication. In contrast, the GXGG-2016 strain infection mediated autophagy primarily through activating only the IRE1-XBP1 pathway, and inhibiting this pathway had no significant impact on viral replication. These findings indicate that while PRV infection can induce cellular autophagy by activating ERS, the specific unfolded protein response (UPR) pathways involved differ significantly between strains of varying virulence. This study provides a foundation for understanding the pathogenesis of different PRV strains and developing novel antiviral drugs.

Mammalian orthoreoviruses (MRVs) infect a wide range of animal hosts, yet epidemiological data from southern China remain limited. Here, we performed regional screening of diarrheic cattle in Guangxi, China, from 2023 to 2025. Using L1-targeted RT-PCR, MRV was detected in 8.4% (15/178) of fecal specimens collected across eight prefectures, with a higher detection rate during the cool (autumn–winter) season (October–March). We obtained two complete S1 segments, assigning one Guangxi strain to MRV1 and the other to MRV3. Phylogenetic analysis of L1 sequences indicated a single local lineage nested within a broader multi-host cluster, which was consistent with segment reassortment. σ1 protein alignment revealed a conserved central/C-terminal scaffold and a variable distal head; notably, the MRV3 σ1 sequence contained an extended ~360–390 aa region compared with MRV1. Overall, these findings provide an epidemiological baseline for Guangxi cattle, integrating detection rate, serotype context, and seasonality to support targeted, seasonally informed surveillance and MRV risk assessment.

Brucellosis, a widespread zoonosis, requires reliable diagnostic tools capable of differentiating infected from vaccinated animals (DIVA), particularly with the emergence of DIVA-compatible vaccines such as Brucella abortus A19-ΔVirB12. VirB12, a key immunogenic component of the type IV secretion system, represents a promising serological target for this purpose. In this study, a VirB12-based indirect ELISA (iELISA) was developed and systematically evaluated for the detection of specific antibodies Recombinant VirB12 protein was successfully expressed in Escherichia coli, purified, and used to immunize mice, generating high-titer polyclonal antiserum (1:204,800). The established iELISA demonstrated excellent repeatability (intra- and inter-assay CVs <10%), high analytical sensitivity (detection up to a 1:3200 serum dilution), and high specificity with no cross-reactivity against other common bacterial pathogens. Clinical validation was performed using 244 bovine serum samples, including 37 Brucella-positive, 20 A19-ΔVirB12-vaccinated, and 187 negative samples.The iELISA exhibited superior overall concordance (98.36%) compared with the Rose Bengal Test (RBT, 90.98%) and showed a higher overall accuracy than a commercial cELISA (91.80%). Notably, although the commercial cELISA demonstrated high sensitivity for detecting positive sera, it failed to distinguish vaccinated from infected animals (0% identification rate), whereas the iELISA correctly identified 90.00% of vaccinated sera, highlighting its clear advantage in DIVA-based diagnostics. Collectively, this assay represents a practical, reliable, and DIVA-compatible diagnostic tool with strong potential for large-scale brucellosis surveillance and control programs.

Getah virus (GETV), an arthropod-borne alphavirus, has emerged as a significant pathogen responsible for reproductive failure and systemic disease in swine, incurring substantial economic losses within the livestock industry and presenting a potential zoonotic threat. Currently, no approved vaccines or antiviral therapies exist. Here, we employed reverse genetics to engineer a recombinant GETV (rGECNLuc) expressing a Nanoluciferase (NLuc) reporter gene. The reporter was inserted at the C-terminus of the viral capsid (Cap) protein via a T2A self-cleavage peptide to facilitate co-translational release. In vitro characterization demonstrated that rGECNLuc replicated efficiently in BHK-21 and Vero cells, with plaque morphology and multi-step growth kinetics comparable to the parental virus, rGETV-GX. The NLuc insertion was genetically stable over six consecutive passages without deletion. In vivo assessment in a 10-day-old suckling mouse model revealed a significant attenuation of virulence for rGECNLuc compared to the parental strain, despite efficient viral replication in joints, lungs, and brain tissues. Using in vivo imaging (IVIS), we established a strong positive correlation between bioluminescent signal intensity and viral load in 3-week-old and 5-week-old mice. Signals disseminated systemically from the inoculation site, with pronounced enrichment in joints and lungs, and exhibited greater intensity in younger animals. Collectively, rGECNLuc enables real-time, non-invasive visualization of GETV infection and provides a tractable platform for quantifying viral dissemination, tissue tropism, and antiviral activity in vivo.

Porcine astrovirus (PAstV) is globally prevalent in swine and is associated with diarrhea and encephalitis in piglets, posing a threat to porcine health. However, its pathogenic mechanisms remain poorly understood. This study used the PAstV1-GX1 strain to infect PK-15 cells, revealing that the virus induces significant apoptosis, with late apoptotic cells reaching 41.2% at 24 hours post-infection. The infection activates caspase-9 and caspase-3, but not caspase-8, and causes mitochondrial damage, indicating apoptosis via the mitochondrial pathway. The apoptosis inhibitor Z-VAD-FMK reduced viral replication, while apoptosis inducer ABT-263 enhanced it at later stages. The nsP1a/3 protein, which interacts with MAVS and localizes to mitochondria, was identified as key in inducing apoptosis. Its 3C-like serine protease domain likely mediates this interaction. Knocking down MAVS reduced apoptosis and increased early-stage replication but decreased it later. Overexpressing MAVS increased apoptosis and decreased replication. Furthermore, we observed that the expression of nsP1a/3 resulted in the cleavage of MAVS and the suppression of the type I interferon (IFN) response. Notably, treatment with Z-VAD-FMK did not influence nsP1a/3-mediated MAVS cleavage or type I IFN inhibition, suggesting that the induction of apoptosis and MAVS cleavage are distinct processes. By employing site-directed mutagenesis to substitute alanine for the catalytic triad residues (His459, Asp487, and Ser549) of the 3C-like serine protease, we significantly reduced the ability of nsP1a/3 to induce apoptosis, cleave MAVS, and suppress the type I IFN response, underscoring the essential role of protease activity in these functions. Furthermore, the use of a serine protease inhibitor markedly decreased PAstV replication. These findings provide significant insights into the pathogenesis of PAstV and establish a foundation for the development of novel antiviral therapies.

The PA gene of the 2009 pandemic H1N1 (pdm09/H1N1) lineage is derived from avian influenza virus (AIV). Together with other polymerase subunits, it enhances the adaptation of avian-origin influenza virus to mammals. However, the functional region of the PA protein remains unclear. Using reverse genetics, we mapped functional domains of pdm09/H1N1 PA in an avian-origin H9N2 canine influenza virus (CIV) background. The N-terminal 169-252 region conferred high intrinsic polymerase activity yet failed to support efficient viral replication alone, revealing a dissociation between catalytic potential and replicative fitness. In contrast, the 85-168 region enabled robust replication despite lower activity. Importantly, synergy between the 169-252 region and its cognate pdm09/H1N1 NP drove high polymerase activity and replication. This pairing also enhanced viral growth in vitro, increased viral titers in mouse lungs and nasal turbinate, and induced pulmonary damage. Mechanistically, the 169-252 region interacts with NP, accelerating nuclear import and cRNA synthesis, thereby optimizing viral RNA replication timing. Thus, PA-NP co-evolution is a key driver of mammalian adaptation, moving beyond single mutations and highlighting internal gene compatibility as a determinant of viral fitness and pandemic potential.

Goose astrovirus (GAstV) is a significant pathogen affecting goslings by inducing visceral gout, yet no commercial vaccine is currently available. This study involved the serial passaging of the GAstV-GXNN strain in LMH cells to investigate alterations in viral replication, genomic stability, and pathogenicity, as well as to assess the potential of a vaccine candidate. The findings indicated that the viral titer increased progressively with each passage, reaching 107.35 TCID50/mL by the 120th passage (GAstV-GXNNP120). Whole-genome sequencing revealed the presence of 6, 19, 26, and 28 nucleotide mutations at the 30th, 60th, 90th, and 120th passages, respectively. Pathogenicity assays demonstrated a reduction in virulence with successive passages, culminating in the complete attenuation of GAstV-GXNNP120, which did not induce clinical signs or lesions in one-day-old goslings. Following five successive passages in goslings, the attenuated strain exhibited stable genetic characteristics without any reversion to virulence. Goslings aged one day, inoculated with GAstV-GXNNP120 at dosages ranging from 102.0 to 105.0 TCID50, developed neutralizing antibodies by the third day post-vaccination. Antibody levels increased in a dose-dependent manner, peaking at day 21 and remaining elevated through day 42. Challenge experiments utilizing the virulent GAstV-GXNN strain revealed that groups vaccinated with doses of 103.0 TCID50 and above achieved complete protection. These groups exhibited no clinical symptoms or pathological damage post-challenge, and both tissue viral loads and virus shedding levels were significantly reduced compared to the control group. Consequently, the minimum effective vaccination dose was established at 103.0 TCID50. These results provide a crucial foundation for the development of a live attenuated GAstV vaccine.

Porcine astrovirus (PAstV) is an important and widespread pathogen in swine, linked to diarrheal outbreaks and extraintestinal disease. How PAstV enters host cells has remained unclear, and no cellular factor has been defined for PAstV entry. Here, a genome-wide CRISPR-Cas9 loss-of-function screen in porcine epithelial cells identifies Annexin A1 (ANXA1) as a host factor that facilitates PAstV entry. Genetic ablation or pharmacological/antibody blockade of ANXA1 reduces binding, lowers early viral RNA and capsid signals, and delays the rise of progeny, whereas re-expression restores susceptibility. Biochemical assays and surface plasmon resonance indicate a direct interaction between ANXA1 and the acidic C-terminal domain of the PAstV ORF2 capsid protein, and imaging shows ANXA1 co-localizes with incoming particles at the cell surface and supports attachment and uptake. Loss of ANXA1 does not alter infection by the non-astrovirus panel tested, indicating selectivity for PAstV under our conditions. Notably, infection is reduced but not abolished in ANXA1-deficient cells, consistent with additional entry factors acting alongside ANXA1. These findings position ANXA1 as an entry cofactor for PAstV and provide a mechanistic basis to refine models of astrovirus host-cell recognition.

The N-linked glycosylation of alphavirus envelope proteins plays critical roles in glycoprotein folding, host-receptor interactions, immune evasion, and pathogenicity. Getah virus (GETV) has two putative N-linked glycosylation sites (N-200 and N-262) in the E2 and one (N-141) in E1. We generated seven glycosylation-deficient mutants and evaluated their fitness across mammalian cells, mosquitoes, and mouse models. Loss of glycosylation at E2 N-262 or E1 N-141 enhanced in vitro replication and replication efficiency in mosquitoes, while E2 N-200 glycosylation-deficient mutants retained parental replication capacity in vitro but exhibited accelerated mosquito colonization. Despite these gains, glycan loss reduced viral adsorption/entry in selected settings and decreased measurable virion binding to MXRA8 and LDLR in vitro, while single-site E2 glycan mutants exhibited increased heparin sensitivity/affinity, indicating altered utilization of glycosaminoglycan attachment pathways. In vivo, all mutants remained lethal in 3-day-old mice but showed age-dependent attenuation in 10-day-old mice. Notably, E1 N141-deficient mutant induced no clinical symptoms and exhibited reduced viremia and tissue viral loads. Glycan ablation increased susceptibility to neutralization without impairing induction of neutralizing antibodies. Strikingly, E2 mutants rapidly reacquired glycosylation during in vivo replication, indicating strong evolutionary selection for these sites. Together, our data support an evolutionary trade-off in which GETV envelope glycans-particularly the epidemic-lineage-associated E2-N262 glycan-optimize overall fitness by balancing replication/transmission efficiency with humoral immune evasion.

Eurasian avian-like H1N1 influenza A viruses that have reassorted with the 2009 pandemic H1N1 virus pose a potential public health threat. We previously reported that synergistic mutations in HA and NS genes enhanced the virulence of a mouse-adapted EA H1N1 canine influenza virus (CIV), yet the underlying mechanisms remained unclear. Here, we demonstrate that the adaptive mutations NS1-A53D and NEP-R42K synergistically promote viral replication by enhancing viral ribonucleoprotein (vRNP) nuclear export, while NEP-R42K alone boosts polymerase activity. Critically, we uncover a novel synergy between HA mutations (N198D and A227E) and NS1-A53D, but not NEP-R42K, in facilitating early viral entry, including attachment and internalization. Mechanistically, NS1-A53D significantly enhances HA protein stability without altering receptor binding specificity, thereby compensating for the intrinsic antagonism between the two HA mutations that impairs receptor binding affinity. These findings reveal that HA and NS genes cooperate through distinct but complementary mechanisms: NS1-A53D stabilizes HA to promote entry, while NS1-A53D and NEP-R42K collaboratively enhance vRNP export, collectively driving mammalian adaptation and pathogenicity.

Goose astrovirus (GAstV) is a major pathogen associated with gout in goslings. Currently, no effective therapeutics or vaccines are available against GAstV infection, and disease control primarily relies on strict biosecurity measures. Therefore, the development of rapid and reliable diagnostic methods is critical for clinical management and reducing the impact of the virus. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of GAstV was developed. The N-terminal region (amino acids 130–401) of the GAstV capsid protein was cloned into the prokaryotic expression vector pET-32a(+) and expressed in Escherichia coli BL21 cells. The recombinant protein, designated GAstV-Cap-N, was purified and used as the coating antigen in the ELISA. Following systematic optimization of key parameters, the established indirect ELISA exhibited high specificity, sensitivity, and reproducibility. Comparative analysis with Western blot demonstrated a concordance rate of 87.5%. The assay was subsequently applied to test 228 clinical serum samples from geese, revealing an overall seropositivity rate of 38.19%. These findings indicate that the developed indirect ELISA provides a robust and reliable tool for clinical diagnosis and epidemiological surveillance of GAstV.

The H3N2 subtype of canine influenza virus (CIV) has emerged as a significant pathogen in canine populations since 2006, causing widespread epidemics globally. The hemagglutinin (HA) protein, particularly the HA1 subunit, plays a critical role in viral entry and is a primary target for vaccine development and antiviral therapies. In this study, we analyzed HA structure and rare codons, immunized BALB/c mice with the truncated HA protein, and generated three HA1-specific mAbs: 2A8, 3C4, and 5A3. mAbs 2A8 and 5A3 targeted the conserved epitope 211QTIIP215, while 3C4 recognized the moderately variable epitope 91SNAFS95. Analysis of H3 CIV HA sequences from GISAID revealed that 211QTIIP215 is highly conserved across H3N2 CIV strains, suggesting its potential as a target for broad-spectrum antibodies development, whereas mutations in 91SNAFS95 exhibits moderate sequence variability. These findings support the design of vaccines and diagnostics for H3N2 CIV, advancing our understanding of CIV biology.

Porcine reproductive and respiratory syndrome virus (PRRSV) employs RNA recombination to generate phenotypic variants that evade host immunity, with recombination hotspots frequently observed in the NSP9 gene of field strains. This study investigates the structural determinants of recombination hotspots in the NSP9 gene by integrating bioinformatics analyses and in vitro and in vivo co-infection experiments. We identified three high-frequency recombination hotspots (7492-7624 nt, 7887-7940 nt and 9009-9464 nt) characterized by AU-rich sequences and stem-loop structures. Notably, these hotspot profiles were conserved between field-derived strains and in vitro co-infection models, supporting the use of experimental systems to highlight candidate recombination-prone contexts. To assess the functional role of RNA motifs, synonymous mutations disrupting AU-rich regions or secondary structures were engineered into the 7887-7940 nt hotspot. These mutations significantly reduced recombination rates, underscoring the contribution of local nucleotide composition and structural features to recombination dynamics. Collectively, our findings define sequence/structure contexts that shape PRRSV recombination signatures and provide a mechanistic framework that may inform efforts to improve the genetic stability of live vaccine candidates by reducing recombination propensity while acknowledging that validation in porcine target cells and/or in vivo models is required.

Getah virus (GETV), a mosquito-borne arbovirus of the Alphavirus genus, poses an emerging threat to livestock economies and public health, underscored by its expanding host range and association with recent outbreaks of heightened virulence. While the functional significance of the 3’ untranslated region (3’UTR) in alphavirus biology is recognized, its specific role in GETV remained undefined. Herein, we elucidate the virological functions of the GETV 3’UTR through a reverse genetics approach, generating a panel of viruses with targeted deletions. We demonstrate that the GETV 3’UTR is remarkably plastic, tolerating a consecutive deletion of up to 310 nucleotides while remaining viable. Deletion of conserved repeat sequence elements (RSEs) induced a cell-type-specific replication deficiency in vitro and significantly attenuated virulence in a murine model. A comprehensive deletion mutant (rGETV-KO310) exhibited further impaired replication kinetics in vitro and was profoundly attenuated in vivo, eliciting only transient morbidity with no mortality in both neonatal and weaned mice. Furthermore, this mutant displayed a significant defect in early colonization within mosquito vectors, indicating a role in vector competence. Comparative transcriptomic profiling of knee joints revealed that attenuation correlates with the altered modulation of critical host immune responses, notably the interferon and MAPK signaling pathways. Collectively, these findings establish the GETV 3’UTR as a pivotal regulator of viral fitness, pathogenesis, and transmission. This work provides a foundational rationale for the strategic development of live-attenuated vaccine candidates based on targeted 3’UTR attenuation.

Swine influenza A viruses (swIAVs) undergo frequent reassortment and evolution, posing significant threats to both animal and human health. In this study, we employed reverse genetics to generate five recombinant viruses and evaluated their potential as vaccine candidates. Through pathogenicity evaluation in mice, we identified two promising candidates: an inactivated vaccine (rPR8-JGL16+2) and a live-attenuated vaccine (rPR8-JGL15+3). Both vaccines induced robust hemagglutination inhibition (HI) antibody responses and specific IgG levels against homologous strains. Notably, the attenuated rPR8-JGL15+3 vaccine provided complete protection against both homologous (H1N1) and heterologous (H3N2) viral challenges, with no detectable viral loads in the lungs or nasal turbinate. Although the inactivated rPR8-JGL16+2 vaccine exhibited effective protective efficacy against influenza viruses, it did not fully inhibit the replication of heterologous viruses in mice. Histopathological assessment revealed that rPR8-JGL15+3 immunization prevented significant lung damage, contrasting with the mild pathology observed in rPR8-JGL16+2-vaccinated animals. These findings demonstrate the successful development of novel vaccine candidates against avian-like H1N1 swIAV, with the attenuated vaccine showing promise for broad protection against circulating strains.

Background Porcine reproductive and respiratory syndrome (PRRS) is a widely prevalent disease of reproductive failure of pregnant pigs and respiratory syndromes in pigs of different ages, especially in piglets. The etiological agents include PRRS virus (PRRSV) genotypes 1 (PRRSV-1) and PRRSV-2, whereas their clinical symptoms are similar and hard to differentiate. It is necessary to establish accurate and reliable methods for differential detection of PRRSV-1 and PRRSV-2.Methods Two pairs of specific primers and probes were designed basing on the PRRSV-1 and PRRSV-2 ORF6 gene. The reaction conditions and procedures of the duplex crystal digital PCR (cdPCR) were optimized. The specificity, sensitivity, and repeatability of the developed assay were evaluated. The application of the developed assay was assessed by testing 2,185 clinical tissue samples.Results The results indicated that the concentration of the templates and their Ct values had good linear relationship with R2 of 0.998. This method could specifically detect PRRSV-1 and PRRSV-2, without cross-reaction with other swine viruses. The limits of detection (LODs) of the assay were 4.507 copies/reaction and 5.607 copies/reaction for PRRSV-1 and PRRSV-2, respectively, which was approximately 30 times more sensitive than that of the duplex real-time quantitative PCR (qPCR). The repeatability test showed that the intra- and inter-assay coefficients of variation (CVs) were 0.74%-0.93% and 0.63%-1.62%, respectively. This method was validated by testing 2,185 clinical samples from Guangxi Province in South China, and the positivity rates of PRRSV-1 and PRRSV-2 were 2.20% (48/2,185) and 23.43% (512/2,185), respectively. The coincidence rates of the developed assay with the qPCR assay recommended by the World Organisation of Animal Health (WOAH) were 99.73% and 99.73%, respectively, while with the duplex qPCR developed in this study were 99.82% and 99.77%, respectively.Conclusions These results indicated that a rapid and accurate duplex cdPCR method with high sensitivity and excellent specificity had been successfully developed for the differential detection of PRRSV-1 and PRRSV-2.

Porcine epidemic diarrhea virus (PEDV) remains a major threat to the global swine industry, leading to severe economic losses due to high mortality and morbidity in piglets. Although significant advances have been made in understanding its genetic features, comprehensive studies on the genetic evolution and epidemiology of PEDV remain limited. In this study, we analyzed PEDV N gene sequences obtained from Guangxi during the period of 2020 to 2023, alongside co-infection patterns involving other enteric viruses, including Porcine Rotavirus (PoRV) and Transmissible Gastroenteritis Virus (TGEV). A total of 559 clinical samples were collected from pig farms, followed by RT-PCR testing and sequencing of the PEDV N gene. Phylogenetic, Bayesian, and spatial analyses were employed to investigate the evolutionary dynamics and transmission patterns of PEDV strains. The results revealed a high PEDV positivity rate of 42.75%, with co-infection primarily involving PEDV and PoRV. Notably, novel 27 bp deletions in the N gene were identified, providing new insights into the virus's temporal and spatial evolution. The analysis also revealed key transmission hubs across multiple regions of China. Collectively, these findings enhance our understanding of PEDV's genetic diversity and epidemiology, offering valuable insights for future vaccine development and control strategies.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious pathogen causing near 100% mortality in neonatal piglets, posing a persistent threat to the global swine industry. In this study, we isolated a PEDV strain, 23GXNN-1 (genotype G2c), from an outbreak in Guangxi, China. Two mRNA vaccine candidates were developed: one encoding the wild-type spike (S) protein S-WT and another incorporating proline-stabilized mutations (S-2P, I1076P/L1077P). Evaluations in HEK293T cells and animal models (BALB/c mice, sows, and piglets) revealed that the S-2P LNP-mRNA vaccine induced superior immunogenicity compared to S-WT, generating robust humoral immunity (elevated IgG/IgA titers and neutralizing antibodies) and enhanced cellular immune responses. Notably, maternal immunization with S-2P conferred 100% survival in piglets challenged with virulent PEDV G2c via efficient passive transfer of antibodies through colostrum, outperforming commercial inactivated vaccines. While vaccines provide limited protection to gut microbiota diversity and intestinal barrier integrity during infection, maternally derived antibodies effectively reduce disease severity. This study not only identifies S-2P as a promising candidate for PEDV control, but also underscores the crucial role of maternal immunity in neonatal protection, advancing mRNA-based strategies against enteric coronaviruses.