The classical swine (CS) H1N1 influenza virus, first isolated in 1930, is highly homologous to the 1918 Spanish influenza virus. CS H1N1 virus, which crossed the interspecies barrier to infect humans has become the dominantly prevalent strain in China's pig population, showing a trend of continuous transmission. However, whether subsequent adaptation of CS H1N1 to mammals would increase their pathogenicity toward humans is unknown. To address this, a mouse-adapted (MA) CS H1N1 virus (A/Swine/Guangdong/1/2011[G11-MA]) was generated through serially passaged in mouse lungs, exhibiting increased virulence compared to the wild-type (WT). Further study showed that the G11-MA strain exhibited amino acid mutations in PB2-D740N, PB1-T56I, PA-T97I and HA-K188E, and the combination of PB2-D740N with PA-T97I improved the replication ability in mammalian cells and mice. The G11-MA strain with PB2 and PA (G11/MA PB2PA) group enhanced the viral polymerase activity, with a similar survival rate and weight loss of mice compared to the G11-MA group. Our study demonstrates that the combination of PB2-D740N and PA-T97I plays a key role in the virulence phenotype of CS H1N1 influenza viruses, and provides important information for evaluating the pandemic risk of swine influenza strains.

The nonstructural protein 4 (Nsp4) of porcine reproductive and respiratory syndrome virus (PRRSV), a 3C-like serine protease, functions significantly in viral polyprotein cleavage and immune evasion. We constructed eight hybridoma cell lines stably secreting monoclonal antibodies (mAbs) against the Nsp4 protein in this study. Furthermore, eight novel antigenic epitopes within Nsp4 were identified using a panel of expressed overlapping truncated proteins. Among these, the epitopes recognized by mAbs 10G5 and 1C11, corresponding to amino acid sequences 49SGVGFNQ55 and 123ITEAGELV130, were highly conserved in PRRSV-2, which was applicable for PRRSV-2 strain detection. MAb 8H2 and 10E11 targeted the linear epitope 73WQGVAPK79. Notably, three mAbs targeting epitopes within 123-142aa can simultaneously recognize both PRRSV-1 and PRRSV-2 strains, indicating their potential for broad detection across PRRSV types. Furthermore, mAb 1C11 enabled Nsp4 expression detection during the early stage of PRRSV infection. This mAb also exhibited a blocking effect against PRRSV-positive sera, suggesting the participation of Nsp4 in the humoral immune response of the host early in infection. Additionally, mAb 1C11 could suppress PRRSV replication in vitro. To sum up, the present study provide robust evidence to understand the structural and antigenic properties of the PRRSV Nsp4 protein, providing potential reference for developing Nsp4-specific early antigen detection methods.

Porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus that causes significant economic losses in the swine industry. It continues to pose a substantial threat to global swine production due to the limited efficacy of existing vaccines. Host-directed antiviral strategies targeting cellular pathways represent a promising approach to combat evolving viral strains. On the basis of our previous findings of a strong correlation between cholesterol transport and PEDV entry, we screened a panel of cholesterol-modulating compounds for anti-PEDV activity. We identified U18666A, an inhibitor of the Niemann-Pick C1 (NPC1) cholesterol transporter, as a potent and broad-spectrum inhibitor against multiple PEDV genotypes in both cell lines and porcine intestinal organoids. To validate NPC1 as a relevant pharmacological target, we utilized CRISPR/Cas9 to knock out NPC1 in Huh7 cells, which significantly inhibited PEDV infection. Conversely, forced expression of NPC1 in knockout cells partially restored viral infectivity. Using both authentic PEDV and PEDV pseudoviruses, we demonstrated that NPC1 facilitates the internalization of PEDV entry. Furthermore, co-immunoprecipitation and structural modeling revealed a direct interaction between NPC1 and the membrane-fusion subunit (S2) of the PEDV spike protein, as well as a role for NPC2 in this process, indicating that the NPC1/NPC2 cholesterol transport complex participates directly in viral entry. Additionally, exogenous cholesterol supplementation partially mitigated the inhibitory effect of U18666A, underscoring that functional intracellular cholesterol transport, not merely the presence of cholesterol, is required for efficient PEDV entry. Collectively, our findings elucidate a mechanism wherein PEDV co-opts the host NPC1/NPC2 cholesterol transport machinery for efficient internalization, thereby identifying this pathway as a promising target for the development of broad-spectrum host-directed antivirals.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) causes severe enteric disease in swine and continues to impose substantial economic losses on the global pig industry. Understanding the host determinants that govern PEDV entry is therefore critical for elucidating the mechanisms of infection. Here, we show that efficient PEDV entry is closely associated with intact and functional intracellular cholesterol transport in host cells. We identified the cholesterol transporter Niemann-Pick C1 (NPC1) as a critical host factor required for PEDV entry and demonstrated that NPC1 facilitates viral internalization through direct interaction with the S2 subunit of the PEDV spike protein. In addition, coordinated functions of the NPC1/NPC2 cholesterol transport system are involved in this process. By revealing the functional dependence of PEDV entry on host cholesterol trafficking, this study highlights this pathway as a potential target for therapeutic intervention.

Porcine deltacoronavirus (PDCoV) is a newly identified pathogen that can potentially undergo cross-species transmission to threaten the safety of swine and humans. The mechanism by which PDCoV nonstructural protein 14 (nsp14) inhibits the expression of IFN-β is unknown. In this study, we showed that PDCoV nsp14 degrades the MAVS, MyD88 and TRAF3 proteins in host cells by proteasomal and autophagy pathways. PDCoV nsp14 recruits the E3 ubiquitin ligase MARCH8 for catalyzing MAVS, MyD88 and TRAF3 protein ubiquitination. These proteins were recognized and transported to lysosomes by the cargo receptor NDP52 for degradation to inhibit the expression of IFN-β. Furthermore, MAVS, MyD88 and TRAF3 were also found to degrade PDCoV nsp14 by selective autophagy. These results reveal the dual function of selective autophagy in the PDCoV nsp14 and host proteins, which can promote the ubiquitination of viral particles and host antiviral proteins to degrade them both for regulating the relationship between virus infection and host innate immunity.

Integrins are cell surface adhesion molecules. They bridge the intracellular and extracellular environments, enabling bidirectional transmembrane signaling and regulating immune responses. However, it remains unclear whether integrin protein β1 (ITGβ1) is involved in innate immune responses. In our previous study, we demonstrated that porcine epidemic diarrhea virus (PEDV) can induce type I interferon (IFN-I) production. In this study, we observed that ITGβ1 expression is rapidly induced following PEDV infection and further established that PEDV infection primarily promoted ITGβ1 expression through upregulation of the transcription factor c-Myc. We hypothesized that ITGβ1 might be involved in PEDV-induced innate immune responses through IFN-I production. Our investigation revealed ITGβ1 overexpression promotes the phosphorylation and subsequent nuclear translocation of both interferon regulatory factor 3 (IRF3) and NF-κB, thereby enhancing SeV-induced IFN-β promoter activity. Furthermore, we showed that ITGβ1 functions as an activator in the melanoma differentiation-associated protein 5 (MDA5)-mediated IFN-I signaling pathway. More importantly, we demonstrated that ITGβ1 is critically involved in PEDV-induced IFN-I antiviral responses. Mechanistically, ITGβ1 facilitates MDA5 oligomerization by specifically interacting with its caspase activation and recruitment domain (CARD), thereby enhancing dsRNA-recruitment capacity. In summary, the findings of this study indicate that ITGβ1 acts as an activator of the MDA5-dependent IFN-I antiviral innate immune response and positively regulates the MDA5-mediated RIG-I-like receptor signaling pathway.IMPORTANCEPorcine epidemic diarrhea virus (PEDV), an alpha coronavirus, severely impacts newborn piglets, leading to acute manifestations including vomiting, diarrhea, dehydration, and high mortality rates in suckling piglets. These consequences have devastating implications for the global swine industry. Within the host's innate antiviral response, RIG-I-like receptors (RLRs) are critical for the activation of the interferon signaling pathway. Integrin proteins, known for their role in regulating bidirectional signal transduction across the cell membrane, are associated with numerous viral infections. In this study, utilizing PEDV as an infection model, we demonstrated that overexpression of ITGβ1 suppresses PEDV replication, while knockdown of ITGβ1 expression enhances it. Additionally, ITGβ1 significantly augments PEDV-induced type I interferon production in host cells. We further elucidated that ITGβ1 interacts with the 2CARD region of MDA5, promoting MDA5 oligomerization and the transmission of activation signals. These findings establish ITGβ1 as a positive regulatory factor in MDA5-mediated RLR signaling pathway. These findings not only identify ITGβ1 as a novel host antiviral protein against PEDV but also reveal, for the first time, a previously unrecognized function of ITGβ1 in the cellular innate antiviral immune response.

Porcine circovirus type 2 (PCV2) serves as the key pathogen linked to porcine circovirus-associated disease, representing a considerable risk to the worldwide swine industry. A blocking enzyme-linked immunosorbent assay (ELISA) was established to identify antibodies specifically targeting PCV2, employing recombinant Cap protein as the antigen and a monoclonal antibody against PCV2 Cap protein as the detector antibody. Utilizing receiver operating characteristic curve analysis, a cutoff value of 33.8% was determined to distinguish between positive and negative serum samples. The sensitivity and specificity of this blocking ELISA method were reported at 94.7% and 96.1%, respectively. Notably, this approach exclusively identified antibodies for PCV2, showing no cross-reactivity with antibodies related to African swine fever virus (ASFV), Porcine epidemic diarrhea virus (PEDV), Porcine pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus. Both intra-assay and inter-assay coefficients of variation were less than 10%. In a comparison involving 402 porcine serum samples, the agreement rate with a commercial indirect ELISA kit reached 98.76%, with a kappa value of 0.888, reflecting high concordance between the two testing methods. This study showcases the blocking ELISA method as an efficient and standardized approach for serological monitoring of PCV2 in swine populations and for assessing seroconversion in vaccinated pigs. IMPORTANCE:Porcine circovirus type 2 (PCV2) has become recognized as a pathogen of significant economic concern within the swine industry. PCV2 mainly affects the immune systems of pigs, leading to a reduction in lymphocytes and resulting in immune suppression in the affected animals. Co-infection with other porcine pathogens can enhance PCV2 infection and exacerbate porcine circovirus disease. Currently, the kits available for detecting PCV2 antibodies primarily employ indirect enzyme-linked immunosorbent assay (ELISA); however, this method is prone to false positives. In contrast, the blocking ELISA method offers enhanced specificity and provides a more straightforward interpretation of results. Previous studies utilizing blocking ELISA for PCV2 antibody detection have depended on plates coated with purified PCV2 virus, a process that is both technically challenging and time-consuming. Consequently, there is a pressing need to develop a new blocking ELISA method that is more efficient to detect antibodies against PCV2.

African swine fever (ASF), caused by African swine fever virus (ASFV), has inflicted severe economic losses on China’s pig industry. Existing ASFV nucleic acid detection methods struggle to identify infected pigs in the pre-viremic stage, especially for recently emerged recombinant ASFV strains that exhibit delayed clinical symptoms and prolonged virus shedding, posing great challenges to ASF prevention and control. To fit the problem, this study established a TaqMan duplex quantitative polymerase chain reaction (qPCR) assay targeting the ASFV p72 gene and porcine Hp gene for early diagnosis of ASFV infection. The qPCR reaction system (20 μL) and conditions were optimized and showed high sensitivity, with detection limits of 1.42 × 101 copies/μL for Hp and 2.23 × 101 copies/μL for ASFV, as well as excellent specificity and reproducibility. Serum cDNA samples from pigs infected with virulent or recombinant ASFV strains were tested, and the result showed that Hp was detectable as early as 1 day post-infection (DPI), however ASFV remained undetectable until 3DPI. Then cDNA samples from cohabitation infection were tested and 80% samples were Hp-positive, although ASFV test was negative.In conclusion, this duplex qPCR assay for simultaneous detection of Hp and ASFV enables pre-viremia diagnosis of ASF, providing a valuable tool for early screening of ASFV-infected pigs.

Background: Porcine epidemic diarrhea virus (PEDV) is a significant pathogen in swine, causing substantial economic losses worldwide. Despite the availability of existing vaccines, there is a critical need for novel vaccine platforms that ensure robust protection while maintaining safety. Methods: A recombinant replication-deficient vesicular stomatitis virus (VSV) vaccine, rVSV∆G-PEDV-S, was developed by pseudotyping the virus with the spike (S) protein from PEDV. To achieve high-titer pseudotyped rVSV particles, a stable Huh7 cell line expressing the PEDV S protein (Huh7-PEDV-S) was generated. The infectivity and replication capacity of rVSV∆G-PEDV-S were evaluated in PEDV-susceptible cell lines and Huh7-PEDV-S cells. The vaccine’s immunogenicity and safety were assessed in BALB/c mice vaccinated intramuscularly with rVSV∆G-PEDV-S. Results: The pseudotyped rVSV∆G-PEDV-S demonstrated infectivity in PEDV-susceptible cell lines and robust replication in Huh7-PEDV-S cells, while remaining replication-deficient in non-complementary cells. In vaccinated BALB/c mice, the vaccine elicited a strong humoral immune response, characterized by high levels of PEDV S1-specific IgG and neutralizing antibodies. No adverse effects, including weight loss or behavioral changes, were observed in the vaccinated mice, confirming the vaccine’s safety. Conclusions: The rVSV∆G-PEDV-S vaccine represents a promising platform for controlling PEDV outbreaks. Its replication-deficient design and pseudotyping methodology ensure safety and adaptability to emerging PEDV variants. These findings highlight the potential of rVSV∆G-PEDV-S as a safe and effective solution to the ongoing challenges posed by PEDV.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that poses a substantial threat to the global swine industry. However, our current understanding of the host factors crucial for PEDV infection remains limited. To identify these host factors, we conducted a genome-wide CRISPR/Cas9 gene knockout screen using a PEDV-permissive cell line. Our results indicate that the endogenous expression of human interferon-inducible transmembrane protein 3 (IFITM3) enhances PEDV entry and replication. Silencing or eliminating endogenous IFITM3 in Huh7 cells significantly suppressed PEDV entry, whereas reintroducing IFITM3 partially restored susceptibility to PEDV. Overexpression of human IFITM3 or IFITM2, but not IFITM1, in Huh7.5 cells substantially increased PEDV entry and replication. Importantly, our results suggest that human IFITM3 influences PEDV entry at a later stage. Furthermore, the overexpression of porcine IFITM1 significantly enhanced PEDV infection in LLC-PK1 cells, whereas the overexpression of porcine IFITM2/3 did not produce similar effects. Notably, removing the C-terminal 15 amino acids of porcine IFITM2/3 resulted in increased PEDV entry. Coimmunoprecipitation analyses showed that all IFITMs interacted with the PEDV S1 protein, indicating a direct role in the viral entry process. Additionally, porcine IFITM1 colocalized with the PEDV S protein at the cell nuclear periphery and enhanced PEDV infection in porcine small intestinal organoids. Overall, our results suggest that IFITMs are critical in facilitating PEDV entry into cells. Targeting IFITMs may provide a promising strategy for controlling PEDV transmission and developing interventions to mitigate the virus's impact on the swine industry. IMPORTANCE:Understanding the mechanisms underlying porcine epidemic diarrhea virus (PEDV) infection is vital for addressing its significant impact on the swine industry. This study reveals that interferon-inducible transmembrane (IFITM) proteins, particularly human IFITM3 and porcine IFITM1, play crucial roles in facilitating PEDV entry and replication. By elucidating these molecular interactions, the research highlights the potential of IFITMs as therapeutic targets for managing PEDV infections and paves the way for antiviral strategies. Moreover, this research extends beyond PEDV management, underscoring the critical role of host factors in controlling the spread of pathogenic coronaviruses.

Identifying genetic dependencies in human colon cancer could help identify effective treatment strategies. Genome-wide CRISPR-Cas9 dropout screens have the potential to reveal genetic dependencies, some of which could be exploited as therapeutic targets using existing drugs. In this study, we comprehensively characterized genetic dependencies present in a colon cancer organoid avatar, and validated tumor-specific selectivity of select pharmacologic agents. We conducted a genome-wide CRISPR dropout screen to elucidate the genetic dependencies that interacted with select driver somatic mutations. We found distinct genetic dependencies that interacted with WNT, MAPK, PI3K, TP53, and mismatch repair pathways and validated targets that could be exploited as treatments for this specific subtype of colon cancer. These findings demonstrate the utility of functional genomic screening in the context of personalized medicine.

ABSTRACT Porcine epidemic diarrhea virus (PEDV) results in PED, which is an infectious intestinal disease with the representative features of diarrhea, vomiting, and dehydration. PEDV infects neonatal piglets, causing high mortality rates. Therefore, elucidating the interaction between the virus and host in preventing and controlling PEDV infection is of immense significance. We found a new antiviral function of the host protein, RNA-binding motif protein 14 (RBM14), which can inhibit PEDV replication via the activation of autophagy and interferon (IFN) signal pathways. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV nucleocapsid (N) protein through the RBM14-p62-autophagosome pathway. Furthermore, RBM14 can also improve the antiviral ability of the hosts through interacting with mitochondrial antiviral signaling protein to induce IFN expression. These results highlight the novel mechanism underlying RBM14-induced viral restriction. This mechanism leads to the degradation of viral N protein via the autophagy pathway and upregulates IFN for inhibiting PEDV replication; thus, offering new ways for preventing and controlling PED. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) is a vital reason for diarrhea in neonatal piglets, which causes high morbidity and mortality rates. There is currently no effective vaccine or drug to treat and prevent infection with the PEDV. During virus infection, the host inhibits virus replication through various antiviral factors, and at the same time, the virus antagonizes the host’s antiviral reaction through its own encoded protein, thus completing the process of virus replication. Our study has revealed that the expression of RNA-binding motif protein 14 (RBM14) was downregulated in PEDV infection. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV N protein via the RBM14-p62-autophagosome pathway and interacted with mitochondrial antiviral signaling protein and TRAF3 to activate the interferon signal pathway, resulting in the inhibition of PEDV replication.

The emergence of drug resistance to virus(i.e., acyclovir(ACV) to herpesviruses) has been termed one of the common clinical issues,emphasizing the discovery of new antiviral agents. To address it, a genome-wide clustered regularly interspaced short palindromic repeats(CRISPR) screening was performed in mouse haploid embryonic stem cells infected with pseudorabies virus(PRV), an α-herpesvirus causing human and pig diseases. The results demonstrated that type 2 voltage-gated chloride channels(CLC-2) encoded by one of the identified genes, CLCN2, is a potential drug target for anti-herpesvirus therapy. CLC-2 inhibitors, omeprazole(OME) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid(DIDS), can efficiently inhibit infection of multiple herpesviruses in cellulo(i.e., PRV, HSV and EBV), and effectively treat murine herpes simplex encephalitis(HSE). Additionally, DIDS was found to inhibit HSV-1 replication by blocking the PI3K/Akt pathway.Most importantly, both DIDS and OME were able to inhibit ACV-resistant HSV-1 strain infection. The study's findings suggest that targeting host-cell factors such as CLC-2 may be a promising approach to tackling herpesvirus drug resistance. The discovery of CLC-2 as a potential drug target for anti-herpesvirus therapy provides a new direction for the development of novel antiviral agents.

Identifying genetic dependencies in human colon cancer could help identify effective treatment strategies. Genome-wide CRISPR-Cas9 dropout screens have the potential to reveal genetic dependencies, some of which could be exploited as therapeutic targets using existing drugs. In this study, we comprehensively characterized genetic dependencies present in a colon cancer organoid avatar, and validated tumor-specific selectivity of select pharmacologic agents. We conducted a genome-wide CRISPR dropout screen to elucidate the genetic dependencies that interacted with select driver somatic mutations. We found distinct genetic dependencies that interacted with WNT, MAPK, PI3K, TP53, and mismatch repair pathways and validated targets that could be exploited as treatments for this specific subtype of colon cancer. These findings demonstrate the utility of functional genomic screening in the context of personalized medicine.

Due to the extensive genetic and antigenic variation in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), as well as its rapid mutability and evolution, PRRS prevention and control can be challenging. An expeditious and sensitive neutralization assay for PRRSV is presented to monitor neutralizing antibodies (NAbs) in serum during vaccine research. Here, a PRRSV expressing eGFP was successfully rescued with reverse genetics based on the infectious clone HuN4-F112-eGFP which we constructed. The fluorescent protein expressions of the reporter viruses remained stable for at least five passages. Based on this reporter virus, the neutralization assay can be easily used to evaluate the level of NAbs by counting cells with green fluorescence. Compared with the classical CPE assay, the newly developed assay increases sensitivity by one- to four-fold at the early antibody response stage, thus saving 2 days of assay waiting time. By using this assay to unveil the dynamics of neutralizing antibodies against PRRSV, priming immunity through either a single virulent challenge or only vaccination could produce limited NAbs, but re-infection with PRRSV would induce a faster and stronger NAb response. Overall, the novel HuN4-F112-eGFP-based neutralization assay holds the potential to provide a highly efficient platform for evaluating the next generation of PRRS vaccines.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus that poses a serious threat to the global pig industry. Despite extensive efforts, the mechanism underlying virus entry for PEDV remains elusive. In this study, we first identified PEDV-susceptible and non-susceptible cell lines by using PEDV spike pseudotyped vesicular stomatitis virus. Subsequently, we conducted a comprehensive transcriptomic analysis on these cell lines. Through integrating differential expression gene analysis with weighted gene co-expression network analysis, we identified the key pathways that are correlated with the PEDV entry. Our analysis revealed a strong correlation between cholesterol, sterols, and lipid transport with PEDV entry, suggesting a potential role for cholesterol transport in the PEDV entry. For further investigation, we treated Huh7, Vero and LLC-PK1 cells with a cholesterol transport inhibitor, ezetimibe, and observed a significant inhibition of PEDV entry and subsequent viral replication in these cells. Interestingly, pre-treating Huh7 cells with ezetimibe resulted in an increase in the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudoviruses. Moreover, we found that cholesterol could facilitate the entry of PEDV into Huh7 and Vero cells, and this promoting effect can be blocked by ezetimibe. These findings suggest that targeting cholesterol transport specifically inhibits PEDV entry into susceptible cells. Our study offers novel insights into the mechanism of PEDV entry and the development of new therapeutic strategies against this economically important virus.

Table S2. TP53 knockout hESCs resistant drugs

Table S1. FDA approved 127 drugs used in this study

Table S3. Negative selection scores of all genes from MAGeCK test

实验室前期shot-gun质谱结果表明PCSK9在PAM空细胞和PRRSV感染组PAM细胞间存在差异表达.为了深入研究PCSK9与PRRSV间的相互作用机制,本实验在体外合成猪PCSK9基因并克隆至原核表达载体pET-28a中,构建重组表达质粒pET-28a-PCSK9.利用该原核表达系统获得了高效表达的重组蛋白PCSK9(约75 kDa),进一步通过镍柱亲和层析技术纯化重组蛋白PCSK9作为免疫原,皮下免疫5-6周龄的BALB/c小鼠(100 ng/只),获得了1株产生PCSK9特异性单克隆抗体的杂交瘤细胞株(2A2B12).应用此单克隆抗体对猪繁殖与呼吸综合征病毒(PRRSV)感染PAM细胞后的内源性PCSK9变化情况进行了检测.Western blot和IFA结果表明本实验制备的PCSK9单克隆抗体具有良好的特异性,抗体针对的表位位于PCSK9的C端结构域(463-705 aa);此外,Western blot结果表明PRRSV感染可抑制PCSK9蛋白的表达,过表达PCSK9对PRRSV的复制有明显的抑制作用.本研究中研制的抗体可为今后猪源PCSK9与PRRSV的相互作用研究提供工具支持.

African swine fever (ASF) is a contagious infectious disease with high lethality which continuously threatens the global pig industry causing huge economic losses. Currently, there are no commercially available vaccines or antiviral drugs that can effectively control ASF. The pathogen of ASF, ASF virus (ASFV) is a double-stranded DNA virus with a genome ranging from 170 to 193 kb and 151 to 167 open reading frames in various strains, which encodes 150-200 proteins. An effective method of monitoring ASFV antibodies, and specific antibodies against ASFV to promote the development of prevention techniques are urgently needed. In the present study, pK205R of ASFV was successfully expressed in mammalian cells using a suspension culture system. An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified pK205R was established and optimized. The monoclonal antibody (mAb) against pK205R recognized a conservative linear epitope (2VEPREQFFQDLLSAV16) and exhibited specific reactivity, which was conducive to the identification of the recombinant porcine reproductive and respiratory syndrome virus (PRRSV) expressing pK205R. The ELISA method efficiently detected clinical ASFV infection and revealed good application prospects in monitoring the antibody level in vivo for recombinant PRRSV live vector virus expressing the ASFV antigen protein. The determination of the conserved linear epitope of pK205R would contribute to further research on the structural biology and function of pK205R. The indirect ELISA method and mAb against ASFV pK205R revealed efficient detection and promising application prospects, making them ideal for epidemiological surveillance and vaccine research on ASF.