Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea in piglets. The ideal route of protection against PEDV for piglets is through passive (lactogenic) immunity, which is not provided by current inactivated and subunit vaccines on the market. In this study, we investigated whether a DNA vaccine encoding the full PEDV spike protein adjuvanted with cyclo-peptide nanotubes (cPNTs) can provide protection against PEDV through active and passive immunity. For the active immunization experiment, piglets were vaccinated, and the immune response was analyzed, followed by a PEDV challenge test. In a separate experiment, to evaluate the passive (lactogenic) immunity elicited by the cPNTs-adjuvanted DNA vaccine, pregnant sows in a local farm were immunized, and the survival of farrowed piglets was examined. The results showed that, in the active immunization experiment, the DNA vaccine elicited IFN-γ and IL-12 production in piglets. IgA antibodies were detected in the serum, and the expansion of CD4 + and CD8 + T cells was observed. Upon virus challenge, vaccinated piglets remained healthy, gained weight, and showed only mild signs of diarrhea, with minimal virus shedding (Ct value of 33, compared with 16 for the saline-vaccinated control group). For the passive immunity experiment, results show that the DNA vaccine administered orally induced higher levels of IgA in the colostrum of vaccinated sows compared to mock vaccination. The survival rate of the farrowed piglets was higher at 84% for the DNA-oral group compared to that of the mock vaccination group (68%). In conclusion, the cPNTs-adjuvanted DNA vaccine can not only generate protective immunity through direct immunization of piglets but also induce lactogenic immunity in pregnant sows to protect farrowed piglets from PEDV infection.

CD163 not only acts as an essential receptor for porcine reproductive and respiratory syndrome virus (PRRSV) infection but also serves as a barrier to cross-species cellular infection by arteriviruses. Among its domains, the scavenger receptor cysteine-rich 5 (SRCR5) domain of CD163 is functionally indispensable. In this study, two monoclonal antibodies (mAbs), designated 5A and 11D, were developed against the SRCR4-6 region of porcine CD163. The linear epitopes recognized by mAbs 5A and 11D were identified as 546CEGHESHLSLCPVAP560 in SRCR5 and 449WDCKNW454 in SRCR4, respectively. Crystal structural analysis and sequence alignment showed that the 546CEGHESHLSLCPVAP560 epitope resides in the long loop 5-6 of SRCR5, which is a key structural region responsible for ligand binding. Meanwhile, the 449WDCKNW454 epitope is located within a highly conserved region of SRCR4, which confers broad cross-species reactivity. Notably, mAb 5A reduced PRRSV infection, while mAb 11D exhibited no anti-PRRSV activity in target cells. Collectively, this study provides essential molecular tools for exploring CD163-domain-dependent cross-species reactivity and developing anti-PRRSV strategies.

African swine fever (ASF), a highly fatal disease often termed the "number one killer" of pigs, presents clinical symptoms indistinguishable from classical swine fever (CSF), such as fever, diarrhea, and vomiting, complicating on-site differential diagnosis. As both ASF and CSF are notifiable diseases under the World Organisation for Animal Health (WOAH), rapid and accurate identification is crucial for effective outbreak management. In this study, we developed a multicolor lateral flow immunoassay (LFIA) based on latex microspheres (LMs) for the simultaneous detection of antibodies against ASF virus (ASFV) and CSF virus (CSFV). The assay enables visual differentiation within 15 min, with red indicating ASFV antibodies and blue indicating CSFV antibodies. After optimization, the LFIA demonstrated a sensitivity of 1:256, equivalent to that of a commercial ASFV ELISA kit and four-fold higher than that for CSFV (1:64). The assay exhibited high specificity, showing no cross-reactivity with other common swine pathogens and bovine viral diarrhea virus (BVDV). When applied to 180 clinical serum samples and compared with commercial ELISA kits, the LFIA achieved Cohen's kappa values of 0.986 for ASFV and 0.918 for CSFV, indicating excellent agreement. Additionally, intra and interbatch evaluations confirmed its robust repeatability. Overall, the multicolor LM-LFIA offers a rapid, sensitive, specific, and cost-effective tool for point-of-care testing (POCT) of ASFV and CSFV antibodies, holding promise for routine field surveillance and disease control.

Abstract Currently circulating swine influenza viruses (SIVs) mainly include H1N1, H1N2, and H3N2 subtypes. In this study, two G4 genotype Eurasian avian-like (EA) H1N1 SIVs were isolated from 556 samples collected between 2023 and 2026. A systematic analysis was conducted on the two EA H1N1 isolates (FYD30 and YZF69) to assess their pandemic potential. The hemagglutinin (HA) proteins of both H1N1 viruses possessed residues 225E and 228S, indicating enhanced affinity for human-like α-2,6-linked sialic acid receptors, which was confirmed by receptor-binding assays. Polymerase activity tests demonstrated that the two SIVs exhibited significantly higher activity in mammalian cells, relative to avian cells, which is consistent with the efficient replication in mammalian cells. Challenge experiments revealed that both H1N1 caused significant pathogenicity in mice and pigs, with YZF69 exhibited higher virulence than FYD30. The higher virulence of YZF69 may be attributed to its molecular features, including the NP Q357K mutation, and an additional glycosylation site in HA. In conclusion, currently circulating EA H1N1 SIVs have acquired key molecular signatures of mammalian adaptation, exhibit enhanced virulence in mammals, and continue to undergo extensive reassortment driven by international swine trade. These findings highlight the potential pandemic risk of SIVs and underscore the urgent need for strengthened surveillance.

Abstract The cGAS-STING pathway has been widely recognized as a critical DNA-sensing pathway that plays a broad-spectrum antiviral role. Livestock, especially pigs, represents one of the most important meat sources. In this study, we identified a key lysine 61 (K61) of porcine STING (pSTING) that plays an essential role in its degradation and antiviral signaling in a species-specific manner, with K61 as the major lysine of pSTING for K48-linked ubiquitination. After virus infection, pSTING recruits the E3 ligase, RNF5, which specifically assembles a K48-linked ubiquitin chain at K61, thereby mediating pSTING proteasomal degradation and reducing its antiviral activity. Meanwhile, the deubiquitylation of K61 is mediated mainly by deubiquitinase USP20, which enhances the stability and antiviral activity of pSTING. Together, given the relatively few lysine numbers in livestock STINGs and species-specific K61 regulation of pSTING stability and antiviral function, the K61 and its specific regulatory enzymes of pSTING could serve as potential targets for breeding of antiviral pigs and design of antiviral drugs, respectively.

Pigs serve as key "mixing vessels" for influenza A viruses, playing a critical role in cross-species transmission, while the H3N2 subtype represents an important lineage within the swine influenza virus (SIV) family. In this study, a novel reassortant H3N2 SIV strain, designated A/Swine/Jiangsu/YZ07/2024, was isolated from pigs exhibiting clinical symptoms in Northern Jiangsu, China during epidemiological survey. Genetic analysis revealed that the virus is a complex reassortant, with the internal genes (M, NP, PB1, PB2, PA) originated from the 2009 pandemic H1N1 lineage, the NS gene exhibiting a North American triple reassortant origin (human-avian-swine origin), and the HA and NA genes belonging to the human-like lineage. Although neither the rescued virus nor its parental strain could replicate effectively in chicken embryos and chicken cells, both demonstrated efficient replication in mammalian cells, reflected by the much higher polymerase activity in mammalian versus chicken cells. The key residues of HA protein (190D, 225D and 228S) collectively enhanced the binding preference for human-type α-2,6-linked sialic acid receptors, which was confirmed by receptor binding assays. Furthermore, mouse infection experiments using the rescued H3N2 demonstrated efficient viral replication in nasal turbinates and lung tissues, accompanied by significant pulmonary pathological damage. These findings indicate that the YZ07 strain, through the vast reassortment and accumulation of adaptive mutations, has acquired potential zoonotic risk, underscoring the importance of surveillance of swine influenza viruses.

African swine fever (ASF) is a highly pathogenic disease caused by the African swine fever virus (ASFV) infection, which can affect pigs of all ages and breeds, posing significant threat to the global pig farming industry. The ASFV p30 protein is an early-expressed viral structural protein; however, its function is not fully understood. In this study, the interaction of viral p30 with host TRIM21 was identified. The ectopic TRIM21 inhibited ASFV replication, while knockdown or knockout of TRIM21 promoted ASFV replication. Further, p30 was found to interact with RIG-I-like receptor (RLR) signaling adaptor MAVS, and during ASFV infection, p30-TRIM21-MAVS interacted with each other. Mechanistically, TRIM21 activated the K27 polyubiquitination of MAVS to induce IRF3 mediated type I interferon (IFN) production, whereas p30 counteracted TRIM21 activated MAVS K27 polyubiquitination to evade RLR signaling mediated antiviral IFN induction. In summary, our study revealed a novel function of ASFV p30, and provided new insights into the immune evasion of ASFV.

Feline and canine coronaviruses (FCoVs and CCoVs) are widely prevalent in companion animals. The recent emergence of a highly virulent FCoV-CCoV recombinant virus, FCoV-23, caused a rapid feline infectious peritonitis (FIP) outbreak in Cyprus, which raises a serious concern about the cross-species transmission of CoVs among companion animals. Here, we evaluated the prevalence, transmission and evolution of FCoV and CCoV in the Jiangsu and Zhejiang provinces of China in 2025. A universal RT-qPCR assay for FCoV and CCoV was established and used for the detection of 700 clinical samples, including 501 from cats and 199 from dogs. A total of 92 samples (13.14%, 92/700) were detected as positive, containing 79 positive samples (15.76%, 79/501) from cats and 13 positive samples (6.5%, 13/199) from dogs. Ten complete S genes and two complete genomes were obtained from positive samples. Fragment comparison and phylogenetic analysis identified one CCoV-I sequence from a feline sample and one FCoV-II sequence from a canine sample. Furthermore, recombination analyses not only detected intra-species recombination events but also inter-species cross-over events. Collectively, this study, for the first time, revealed that cross-species transmission and recombination events occurred between FCoV and CCoV among companion animals in the Jiangsu-Zhejiang region of China in 2025.

The innate immune cGAS-STING-IRF3-IFN signaling pathway plays a crucial role in host antiviral defense. While mammalian IRF families encompass IRF1 through IRF9, the involvement of IRF members other than IRF3 in cGAS-STING antiviral signaling transduction and/or regulation remains poorly characterized. Through dual screening systems, we identified the porcine (p) IRF2 as a potent suppressor of porcine cGAS-STING-IFN antiviral signaling. First, we demonstrate that pIRF2 suppresses both cGAS-STING- and TBK1/IKKε/IRF3-activated IFN signaling. Further, we reveal that pIRF2 does not affect STING-activated IRF3 phosphorylation, nor does it impair IRF3 dimerization or nuclear translocation. The inhibitory activity of pIRF2 depends on its DNA binding domain (DBD) but not IRF-associated domain (IAD). Importantly, we find that pIRF2 directly binds the IFN-β promoter and competes IRF3 for binding to IFN-β promoter. In summary, our findings unveil a distinct function of pIRF2 as well as the regulatory mechanism governing porcine cGAS-STING-IFN antiviral function.

Porcine reproductive and respiratory syndrome virus (PRRSV) are responsible for reproductive failure in sows and respiratory diseases in pigs, inflicting significant economic losses worldwide. Due to continuous mutation and recombination, PRRSV exhibit remarkable genetic diversity, necessitating detection methods capable of recognizing PRRSV-2 and/or PRRSV-1 strains. In this study, four monoclonal antibodies (mAbs) targeting the PRRSV-1 nucleocapsid (N) protein were generated, and the precise and novel linear B-cell epitopes were identified as 54PHFPL58, 53KPHFP57, and 59AAEDDIRHH67, respectively. The epitopes 54PHFPL58 and 53KPHFP57 are highly conserved across all major PRRSV-2 lineages and PRRSV-1 subtypes, whereas the epitope 59AAEDDIRHH67 is relatively conserved only in PRRSV-1. Correspondingly, the PRRSV-1 specific mAb 6B4 showed reaction only with PRRSV-1 strains, but not with PRRSV-2 strains. The 6B4 peptide-based ELISA was developed for the detection of PRRSV-1 antibody, which showed an agreement rate of 83.6% with commercial PRRSV Ab ELISA kit in testing 55 clinical samples, indicating the potential of this peptide-based ELISA for preliminary application.

IntroductionThe innate immune cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-interferon (IFN) signaling axis is a cornerstone of antiviral innate immunity in chickens, with chicken interferon regulatory factor 7 (chIRF7) serving as the principal transcription factor downstream chSTING to initiate type I IFN production. Although the chicken interferon regulatory factor (IRF) family consists of eight members, the roles of those beyond chIRF7 in this pathway remain largely unexplored.MethodsSystematic screening was performed to analyze the roles of chicken IRF family members in the cGAS-STING-IFN antiviral signaling. Combined molecular biological and biochemical methods were utilized to explore the action mechanism of chIRF10 as a negative regulator of cGAS-STING-IFN antiviral signaling pathway in transfected cells and gene knockout cells, with or without different virus infections.ResultsThe chIRF10 was identified as a potent negative regulator of chicken cGAS-STING-IFN antiviral signaling. Mechanistically, chIRF10 mediated suppression required its IRF associated domain (IAD) but not its DNA binding domain (DBD). chIRF10 inhibited IFN activation triggered by cGAS-STING, as well as by the downstream kinases TBK1/IKKε and the transcription factor IRF7. Importantly, chIRF10 physically interacted with chIRF7 via its IAD, impairing chIRF7 dimerization and activation.DiscussionCollectively, these findings unveil a novel immunoregulatory function of chIRF10 and delineate a previously unrecognized mechanism fine-tuning the chicken cGAS-STING-IFN antiviral pathway, offering potential insights for developing interventions against avian viral diseases.

BACKGROUND:The zoonotic parasite Plasmodium knowlesi is an increasing cause of human malaria in Southeast Asia. We aimed to develop a P. knowlesi induced blood-stage malaria (IBSM) model to facilitate study of parasite biology, host responses to infection, and activity of antimalarial treatments. METHODS:We manufactured and characterised a P. knowlesi parasite bank using the P. knowlesi YH1 strain adapted to grow in human serum. This P. knowlesi bank was evaluated in an IBSM study involving four adults intravenously inoculated with P. knowlesi-infected erythrocytes using a dose escalation strategy (10-fold increases from ∼2,800 parasites). Parasitaemia was monitored by qPCR. All participants received curative treatment with artemether-lumefantrine. Endpoints included safety and infectivity of the P. knowlesi bank. RESULTS:Two of the four participants developed detectable parasitaemia. Participant two (administered ∼28,000 parasites) had detectable parasites (∼6 parasites/mL) immediately prior to protocol-defined treatment on day 21. Participant three (administered ∼280,000 parasites) developed detectable parasites on day 17, peaking at 202 parasites/mL just after protocol-defined treatment on day 24; the parasite multiplication rate over 32 hours (PMR32) was 2.33 (95% CI 1.66-3.25). Participant four (also administered ∼280,000 parasites) did not become infected. Adverse events were mostly mild and unrelated to the challenge agent. CONCLUSIONS:A GMP-grade P. knowlesi YH1-HS parasite bank was successfully manufactured and shown to establish patent infection in humans. The finding that only one of two participants became infected at the highest inoculum dose highlights the potential for this IBSM model to evaluate factors influencing inter-individual variability in susceptibility to infection.

Manganese ions (Mn2+) are an essential trace element within organisms spanning the entire tree of life. It has reported that Mn2+ exerts strong immunocompetence effects and exhibits antiviral effects against various human and animal viruses, including DNA and RNA viruses. Recently, Mn2+ has been found to be involved in the activation of the innate immune DNA-sensing cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway and subsequent antiviral function. However, the antiviral mechanism of Mn2+ remains unclear. In the current study, the results suggest that the cGAS-STING pathway is essential for Mn2+ to promote interferon (IFN) signaling, but it is not essential for triggering antiviral functions. After knocking out the STING or interferon regulatory factor 3 (IRF3) gene, Mn2+ still retains its antiviral activity against herpes simplex virus type 1 (HSV-1) and vesicular stomatitis virus (VSV). Furthermore, the results from transcriptomic analysis indicate that Mn2+ can induce a significant change in the apoptotic process in STING-/- 3D4/21 cells. Mn2+ can induce cell apoptosis through the oxidative stress pathway, and inhibiting the apoptotic signal could suppress Mn2+-mediated antiviral activity in STING-/- 3D4/21 cells. Additionally, dual knockout of IRF3 and caspase3, resulting in concurrent loss of IFN and apoptotic signals, eliminates the antiviral effects of Mn2+. In summary, the current study suggests that Mn2+ could exert antiviral effects not only through the cGAS-STING-IFN pathway but also via the reactive oxygen species (ROS)-apoptosis pathway.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is an important pathogen that seriously endangers pig breeding, causing significant economic losses to the global swine industry. Our previous study found that the DNA sensing innate cGAS-STING signaling pathway plays an important role in inducing interferon (IFN) upon PRRSV infection and inhibition of PRRSV replication. However, the mechanism underlying immune evasion by PRRSV remains unclear. In the current study, we found that PRRSV non-structural protein 5 (Nsp5) strongly inhibits the cGAS-STING-IFN antiviral response. Furthermore, we found that Nsp5 interacts with STING, blocking STING transport from the ER to the Golgi apparatus and interfering with STING recruitment of TBK1/IKKε/IRF3. Finally, we demonstrated that the Nsp5 36-47 and 58-67 amino acid regions are critical for inhibiting STING activity and PRRSV replication. This study describes a novel mechanism by which PRRSV suppresses the host innate antiviral response and has implications for our understanding of PRRSV pathogenesis.

NADC34-like porcine reproductive and respiratory syndrome virus 2 (NADC34-like PRRSV-2) has become prevalent in Chinese swine herds. However, current commercial vaccines only provide limited cross-protection against NADC34-like PRRSV-2 infection. In our previous study, we developed the infectious clone (rBJ1805-2) of a non-recombinant NADC34-like PRRSV-2 BJ1805-2 isolate and identified the GP2a 91/97/98 amino acid substitutions (from "TMF" to "VVL" pattern) as a sufficient and necessary determinant for its Marc-145 cell tropism. In this study, the non-recombinant Marc-145-adapted NADC34-like mutant (rBJ-VVL) was serially passaged in Marc-145 cells for 30 times to generate rBJ-VVL-P30. The rBJ-VVL-P30 reached significantly increased titer in Marc-145 cells but significantly reduced replication in PAMs when compared with the parental virus. Reverse genetics and genome sequencing confirmed that 13 amino acid substitutions are responsible for the changed replication efficacy of rBJ-VVL-P30. Considering that NADC34-like PRRSV-2 recombinants are predominant in China, we evaluated the efficacy of rBJ-VVL-P30 strain against a recombinant NADC34-like SDLY23-1742 isolate sharing 83.93 % genome similarity. Piglets that inoculated with rBJ-VVL-P30 did not present fever or any clinical signs. More importantly, when challenged with the SDLY23-1742 recombinant at 42 days post inoculation (dpi), neutralizing antibodies (nAbs), T follicular helper (Tfh) cells and PRRSV-specific IFN-γ secreting cells (IFN-γ-SC) could be induced in rBJ-VVL-P30 pre-immunized pigs conferring cross-protection. Our findings support that the rBJ-VVL-P30 strain is a non-recombinant NADC34-like PRRSV-2 vaccine candidate that can provide satisfied protection against the recombinant NADC34-like PRRSV-2 isolates.

African swine fever (ASF) is caused by the African swine fever virus (ASFV); infection in domestic pigs and wild boars leads to a highly contagious, hemorrhagic disease. The p54 protein is encoded by the ASFV E183L gene and is an important structural protein located on the inner envelope of the virus. It is involved in processes of virus assembly, apoptosis induction, and neutralizing antibody production. In this study, three specific monoclonal antibodies (mAbs) against ASFV p54 protein were generated, namely 6B11, 3E3, and 3C10, from mice who were immunized with recombinant prokaryotic p54-truncated protein. Three novel linear B cell epitopes, recognized by the mAbs, were revealed: 60AAIEEEDIQFINP72, 128MATGGPAAAPAAASAPAHPAE148, and 163MSAIENLRQRNTY175. The epitopes 60AAIEEEDIQFINP72 and 163MSAIENLRQRNTY175 were highly conserved in genotype I and II ASFV strains. In addition, the epitope peptide ELISA can be used for the detection of ASFV antibodies. Our work provides new insights for p54 antigenicity and an alternative tool for serological diagnosis of ASF.

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) isolates share a restricted cellular tropism. Marc-145 cells derived from African green monkey are one of the few cell lines supporting PRRSV propagation in vitro and are commonly used for PRRS vaccine development. However, currently prevalent PRRSV isolates display different Marc-145 cell tropism while the exact determinant is not clarified yet. In this study, we identified for the first time that the 91/97/98 amino acid (aa) substitutions in GP2a of PRRSV play critical roles in determining Marc-145 adaptation. Specifically, multiple series of chimeric viruses were constructed based on four PRRSV infectious clones including Marc-145 adaptive HP-PRRSV-2 strain and Marc-145 non-adaptive NADC34-like PRRSV-2, NADC30-like PRRSV-2, and PRRSV-1 strains. The GP2a 91/97/98 aa substitutions are a sufficient and necessary determinant in NADC34-like and NADC30-like PRRSV-2, a sufficient but not necessary determinant in HP-PRRSV-2, a necessary but not sufficient determinant in PRRSV-1, respectively. In addition, the GP2a substitutions also influenced PRRSV infectivity in PAMs and piglets. Noticeably, the GP2a substitutions did not significantly affect the levels of neutralizing antibodies, porcine T follicular helper (Tfh) cells, and PRRSV-specific IFNγ secreting cells. Overall, our results not only provide new insights into PRRSV tropism and infectivity but also will facilitate PRRS vaccine development. IMPORTANCE Prevalent PRRSV isolates present different cell tropisms in vitro . Clarifying the exact determinant of PRRSV tropism is crucial for PRRSV isolation and vaccine development. By constructing chimeric viruses based on four representative PRRSV infectious clones, we identified for the first time that the 91/97/98 amino acid substitutions in GP2a play critical but distinct roles in determining Marc-145 cell tropism for different PRRSV strains. The GP2a 91/97/98 amino acid substitutions also affect PRRSV infectivity in PAMs and piglets but do not influence immune responses. This study not only deciphers an exact determinant of PRRSV tropism and infectivity but also has guiding significance for PRRS vaccine development.

African swine fever virus (ASFV) causes a highly contagious and lethal hemorrhagic disease and significantly threatens the pig industry. There is no commercially effective vaccine available currently, making the detection of ASFV critical for control and prevention. Previously, we established the CRISPR-LbCas12a and LwCRSIRP-Cas13a visual detections of ASFV, separately, targeting the structural p17 gene D117L. In this study, we performed the parallel detections of ASFV based on the conserved viral protease gene S273R using CRISPR-LbCas12a and CRISPR-LbuCas13a systems. Our results showed that both systems are able to specifically detect ASFV as low as two copies of the S273R gene, and effectively detect clinical samples with minimal DNA purification. The work promotes CRISPR-Cas systems for the application of on-site detection in the field.

African swine fever (ASF), induced by the African swine fever virus (ASFV), is an acute hemorrhagic disease characterized by high fever, systemic hemorrhages, and elevated mortality. Current diagnostic techniques including PCR and ELISA present limitations in field applications due to requirements for specialized equipment and prolonged processing duration. Therefore, rapid and accurate detection of ASFV has become a key link in ASF prevention and control. This study established a rapid and precise visual diagnostic approach by integrating the CRISPR/AapCas12b system with lateral flow strip (LFS) technology, specifically targeting the B646L gene encoding the major capsid protein p72. The CRISPR/AapCas12b-LFS platform achieved a sensitivity threshold of 6 copies/µL for B646L gene detection, completing analysis within an hour. Validation study confirmed exceptional specificity against common porcine pathogens including PRRSV, CSFV, PRV, PPV4, and PCV3. The developed assay demonstrated complete concordance with real-time PCR results when analyzing 34 clinical specimens including three heart samples, three liver samples, three spleen samples, three lung samples, three kidney samples, three lymph node samples, five serum samples, five blood samples, and five oral swab samples for ASFV detection. Overall, this method is sensitive, specific, and practicable onsite for ASFV detection, showing a great application potential for monitoring ASFV in the field.

Since the first isolation of the porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) BJEU06-1 strain from a Beijing pig farm in 2006, more and more PRRSV-1 isolates have been identified in China. In this study, we performed the routine detection of PRRSV-1 using 1521 clinical samples collected in 12 provinces/cities from February 2022 to May 2024. Only three lung samples from severely diseased piglets collected in January 2024 were detected as PRRSV-1-positive (0.197%, 3/1521). A PRRSV-1 strain (AHEU2024-2671) was successfully isolated in primary alveolar macrophages (PAMs) but not in Marc-145 cells. Genome sequencing showed that the AHEU2024-2671 isolate shared the highest genome similarity (90.67%) with the SC2020-1 isolate but only 84.01% similarity with the predominant BJEU06-1 strain. Noticeably, the AHEU2024-2671-like isolates not only contained deletions in nsp2 and the GP3-GP4 overlap region, but also contained a unique 6 nt deletion between nsp12 and the ORF2 gene. Furthermore, a genome-based phylogenetic tree supported that the AHEU2024-2671-like isolates form a novel subgroup within subtype 1. Overall, this study not only supported the idea that PRRSV-1 is rapidly evolving in Chinese swine herds, but also pulled the alarm that novel PRRSV-1 isolates with potentially increased pathogenicity might already exist in China, although they are still rarely detected among Chinese pigs.