Mitochondrial ribosomal protein S24 is associated with immunosuppressive microenvironment and cold tumor in lung adenocarcinoma.

Objective: MRPS24 (Mitochondrial Ribosomal Protein S24) belongs to the mitochondrial ribosomal protein family, which participates in the protein synthesis of the mitochondrion. However, the relationship of MRPS24 with lung adenocarcinoma (LUAD) remained unknown. We aimed to identify its immunological and functional mechanisms in LUAD. Methods: The analysis of MRPS24 expression, clinical features, diagnosis, prognosis, function analysis, genetic alteration, copy number variations, methylation, and tumor microenvironment was investigated by the TCGA, UCSC Xena, GEO, HPA, GEPIA, cBioPortal, MethSurv, TIMER, TIMER2.0, and TISIDB databases. Results: MRPS24 was found to be more abundant in LUAD tumor tissue than in normal tissue. High levels of MRPS24 expression were found to be an independent prognostic factor by multivariate analysis. Functional analysis revealed that MRPS24 expression was associated with the immune, cell cycle and methylation. MRPS24 methylation level was inversely linked with its expression (p < 0.001). Patients with low MRPS24 methylation had a worse prognosis than those with high methylation (p < 0.05). In addition, the result revealed that the MRPS24 expression was inversely linked to the immune cell infiltration in LUAD. Finally, the validations of the expression level, prognosis, and immune cell infiltration of MRPS24 were in accordance with our previous results. Conclusions: This study systematically explored that MRPS24 expression was significantly correlated with prognosis, tumorigenesis, genetic alteration, copy number variations, methylation, and immune cell infiltration in LUAD. MRPS24 might be a potential immune-related biomarker in the development and treatment of LUAD, thereby acting as a promising predictor of immunotherapy response in LUAD.

Identification of Three Novel Linear B-Cell Epitopes in Non-Structural Protein 3 of Porcine Epidemic Diarrhea Virus Using Monoclonal Antibodies.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic swine coronavirus that causes diarrhea and high mortality in piglets, resulting in significant economic losses within the global swine industry. Nonstructural protein 3 (Nsp3) is the largest in coronavirus, playing critical roles in viral replication, such as the processing of polyproteins and the formation of replication-transcription complexes (RTCs). In this study, three monoclonal antibodies (mAbs), 7G4, 5A3, and 2D7, targeting PEDV Nsp3 were successfully generated, and three distinct linear B-cell epitopes were identified within these mAbs by using Western blotting analysis with 24 truncations of Nsp3. The epitope against 7G4 was located on amino acids 31-TISQDLLDVE-40, the epitope against 5A3 was found on amino acids 141-LGIVDDPAMG-150, and the epitope against 2D7 was situated on amino acids 282-FYDAAMAIDG-291. Intriguingly, the epitope 31-TISQDLLDVE-40 recognized by the mAb 7G4 appears to be a critical B-cell linear epitope due to its high antigenic index and exposed location on the surface of Nsp3 protein. In addition, bioinformatics analysis unveiled that these three epitopes were highly conserved in most genotypes of PEDV. These findings present the first characterization of three novel linear B-cell epitopes in the Nsp3 protein of PEDV and provide potential tools of mAbs for identifying host proteins that may facilitate viral infection.

Inhibition of pseudorabies virus replication via upregulated interferon response by targeting 7-dehydrocholesterol reductase.

Pseudorabies virus (PRV) is an alpha-herpesvirus capable of infecting a range of animal species, particularly its natural host, pigs, resulting in substantial economic losses for the swine industry. Recent research has shed light on the significant role of cholesterol metabolism in the replication of various viruses. However, the specific role of cholesterol metabolism in PRV infection remains unknown. Here, we demonstrated that the expression of 7-dehydrocholesterol reductase (DHCR7) is upregulated following PRV infection, as evidenced by the proteomic analysis. Subsequently, we showed that DHCR7 plays a crucial role in promoting PRV replication by converting 7-dehydrocholesterol (7-DHC) into cholesterol, leading to increased cellular cholesterol levels. Importantly, DHCR7 inhibits the phosphorylation of interferon regulatory factor 3 (IRF3), resulting in reduced levels of interferon-beta (IFN-ß) and interferon-stimulated genes (ISGs). Finally, we revealed that the DHCR7 inhibitor, trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride (AY9944), significantly suppresses PRV replication both in vitro and in vivo. Taken together, the study has established a connection between cholesterol metabolism and PRV replication, offering novel insights that may guide future approaches to the prevention and treatment of PRV infections.

Pathogenicity and immunogenicity of a quadruple gene-deleted pseudorabies virus variant as a vaccine candidate.

Since late 2011, the PRV variants have emerged in China, characterized by the increased virulence. The traditional attenuated vaccines have proven insufficient in providing complete protection, resulting in substantial economic losses to swine industry. In this study, a vaccine candidate strain, ZJ01-ΔgI/gE/TK/UL21, carrying the quadruple gene deletion was derived from the previously generated three gene-deleted virus ZJ01-ΔgI/gE/TK. As anticipated, piglets inoculated with ZJ01-ΔgI/gE/TK/UL21 exhibited normal body temperatures and showed no viral shedding, consistent with the observations from piglets treated with ZJ01-ΔgI/gE/TK. Importantly, a significant higher level of interferon induction was observed among piglets in the ZJ01-ΔgI/gE/TK/UL21 group compared to those in the ZJ01-ΔgI/gE/TK group. Upon challenge with the PRV variant ZJ01, piglets immunized with ZJ01-ΔgI/gE/TK/UL21 exhibited reduced viral shedding compared to the ZJ01-ΔgI/gE/TK group. Furthermore, piglets vaccinated with ZJ01-ΔgI/gE/TK/UL21 exhibited minimal pathological lesions in brain tissues, similar to those in the ZJ01-ΔgI/gE/TK group. These results underscore the potential of ZJ01-ΔgI/gE/TK/UL21 as a promising vaccine for controlling PRV infection.

Vidofludimus inhibits porcine reproductive and respiratory syndrome virus infection by targeting dihydroorotate dehydrogenase.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection has caused huge economic losses in global swine industry over the last 37 years. PRRSV commercial vaccines are not effective against all epidemic PRRSV strains. In this study we performed a high-throughput screening (HTS) of an FDA-approved drug library, which contained 2339 compounds, and found vidofludimus (Vi) could significantly inhibits PRRSV replication in Marc-145 cells and primary porcine alveolar macrophages (PAMs). Compounds target prediction, molecular docking analysis, and target protein interference assay showed that Vi interacts with dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in the de novo pyrimidine synthesis pathway. Furthermore, PRRSV infection was restored in the presence of excess uridine and cytidine which promote pyrimidine salvage, or excess orotate which is the product of DHODH in the de novo pyrimidine biosynthesis pathway, thus confirming that the antiviral effect of Vi against PRRSV relies on the inhibition of DHODH. In addition, Vi also has antiviral activity against Seneca virus A (SVA), encephalomyocarditis virus (EMCV), porcine epidemic diarrhea virus (PEDV), and pseudorabies virus (PRV) in vitro. These findings should be helpful for developing a novel prophylactic and therapeutic strategy against PRRSV and other swine viral infections.

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components.

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

Novel Epitope Mapping of African Swine Fever Virus pI215L Protein Using Monoclonal Antibodies.

The African swine fever virus (ASFV) is one of the most important pathogens that causes huge damage to worldwide swine production. The pI215L protein is found within the virion and expressed at a high level in infected porcine alveolar macrophages (PAMs), indicating a possible role of pI215L protein in ASFV detection and surveillance. In the present study, female BALB/c mice (5-6-week-old) were immunized with rpI215L protein, and six hybridomas, 1C1, 2F6, 2F10, 3C8, 5E1 and 5B3, steadily secreted anti-pI215L monoclonal antibodies (mAbs). Among them, 1C4, 5E1, and 5B3 had the IgG1 isotype with a Lambda light chain, 2F10 and 3C8 had the IgG1 isotype with a Kappa light chain, and 2F6 had the IgG2a isotype with a Kappa light chain. Western blot showed a good reactivity of the six mAbs against ASFV. Eight truncated polypeptides were produced for epitope mapping. Two novel B cell epitopes, 67LTFTSEMWHPNIYS80 and 167IEYFKNAASN176, were identified by the mAbs. Further analysis revealed that 2F6 mAb could be widely used in ASFV surveillance and 5B3 mAb might serve as a tool in the distinguishment of different ASFV genotypes. This study provides tools of monoclonal antibodies for further study of I215L function and contributes to the development of serological diagnosis and vaccine research.

SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy.

IMPORTANCE: Senecavirus A (SVA) is an emerging picornavirus associated with vesicular disease, which wide spreads around the world. It has evolved multiple strategies to evade host immune surveillance. The mechanism and pathogenesis of the virus infection remain unclear. In this study, we show that SERPINB1, a member of the SERPINB family, promotes SVA replication, and regulates both innate immunity and the autophagy pathway. SERPINB1 catalyzes K48-linked polyubiquitination of IκB kinase epsilon (IKBKE) and degrades IKBKE through the proteasome pathway. Inhibition of IKBKE expression by SERPINB1 induces autophagy to decrease type I interferon signaling, and ultimately promotes SVA proliferation. These results provide importantly the theoretical basis of SVA replication and pathogenesis. SERPINB1 could be a potential therapeutic target for the control of viral infection.

Porcine reproductive and respiratory syndrome virus-mediated lactate facilitates virus replication by targeting MAVS.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important causative agents in the pig industry worldwide, causing reproductive failure in sows and respiratory problems in growing pigs. Glucose metabolism is a major pathway for energy production and interacts with many cellular processes, such as innate immunity response. It is unclear whether PRRSV infection can use the glucose metabolic pathway to generate immune escape in favor of viral replication. Here, we found that high glucose promotes PRRSV replication and glycolysis, and inhibits poly(I:C)-induced RLR signaling. Conversely, inhibition of the glycolysis pathway significantly promoted poly(I:C)-induced RLR signaling and inhibited PRRSV replication, suggesting that glycolysis promotes PRRSV replication by inhibiting interferon signaling. Furthermore, PRRSV promotes glycolysis to produce lactate, which acts as a key metabolite to promote viral replication by inhibiting RLR signaling by targeting MAVS. And the glycolytic inhibitors targeting HK2 and LDHA in glycolysis could inhibit PRRSV replication. Taken together, these findings suggested that PRRSV infection promotes glycolysis to produce lactate, which targets MAVS to inhibit RLR signaling and thus promote viral replication. Our findings provide an insight into the pathogenesis of PRRSV and offer a theoretical basis for further development of antiviral therapeutic targets.

Identification of new conserved linear B-cell epitopes in the 3AB and 3C protein of Senecavirus A.

Senecavirus A (SVA) is a member of the Picornaviridae family, Senecavirus genus. The outbreak of swine vesicular disease caused by SVA has presented a significant threat to pig husbandry and public health, resulting in substantial economic losses. In this study, recombinant SVA 3AB and 3C proteins were expressed in the prokaryotic system, purified, and utilized to generate eight monoclonal antibodies (mAbs) specific to SVA 3AB or 3C proteins. Three B-cell epitopes recognized by these mAbs were subsequently identified by Western blotting. The mAbs 3G3, 3D6, and 3B7 against 3AB recognize the epitope 90NAYDGPKKNS100; the mAbs 2C10, 2C8, and 2D12 against 3C recognize the epitope 75FTHHGLPTDL85, and the mAbs 3C4 and 4A11 against 3C recognize the epitope 95DQMPARNSRV105. Moreover, all three epitopes are highly conserved in different SVA strains and are exposed on the surface of 3AB or 3C proteins, potentially representing important B-cell epitopes. This study constitutes the first report of SVA nonstructural protein epitopes, which may be beneficial for developing innovative detection methods and vaccines and for investigating the roles of 3AB and 3C in viral replication.

The Papain-Like Protease of Porcine Reproductive and Respiratory Syndrome Virus Impedes STING Translocation from the Endoplasmic Reticulum to the Golgi Apparatus by Deubiquitinating STIM1.

Stimulator of interferon (IFN) genes (STING) was recently pinpointed as an antiviral innate immune factor during the infection of RNA viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), the swine arterivirus, is an enveloped RNA virus which has evolved many strategies to evade innate immunity. To date, the interactive network between PRRSV and STING remains to be fully established. Herein, we report that STING suppresses PRRSV replication through type I interferon signaling. However, PRRSV impedes STING trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, leading to the decreased phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). Furthermore, PRRSV nonstructural protein 2 (Nsp2) colocalizes with STING, blocks STING translocation, and disrupts the STING-TBK1-IRF3 complex. Mechanistically, PRRSV Nsp2 retains STING at the ER by increasing the level of Ca2+ sensor stromal interaction molecule 1 (STIM1) protein. Functional analysis reveals that PRRSV Nsp2 deubiquitinates STIM1 by virtue of its papain-like protease 2 (PLP2) deubiquitinating (DUB) activity. Finally, we demonstrate that loss of STIM1 is associated with an elevated IFN response and restricts PRRSV replication. This work delineates the relationship between PRRSV infection and STING signaling and the importance of papain-like proteases (PLPs) in interfering in this axis. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the family Arteriviridae, is responsible for reproductive disorders in pregnant sows and respiratory problems in piglets, resulting in huge losses in the swine industry worldwide. Of note, PRRSV infection causes immunosuppression, of which the mechanism is not completely understood. Here, we demonstrate for the first time that STING, a protein typically associated with the antiviral response in DNA viruses, plays a critical role in controlling PRRSV infection. However, PRRSV utilizes its encoded protein Nsp2 to inhibit STING activity by blocking its translocation from the ER to the Golgi apparatus. In particular, Nsp2 retains STING at the ER by interacting with and further deubiquitinating STIM1. For this process, the activity of the viral PLP2 DUB enzyme is indispensable. The study describes a novel mechanism by which PLP2 plays a critical role in suppressing the innate immune response against arteriviruses and potentially other viruses that encode similar proteases.

TRIM7 inhibits encephalomyocarditis virus replication by activating interferon-ß signaling pathway.

Tripartite motif-containing protein 7 (TRIM7), the member of tripartite motif (TRIM) family, plays an important role in innate immune responses against viral infection. Among them, the function of TRIM7 in Encephalomyocarditis virus (EMCV) infection has not been reported. Here, we found that TRIM7 inhibited the replication of EMCV through the type I interferon (IFN) signaling pathway. Interestingly, TRIM7 was down-regulated after EMCV infection in HEK293T cells. Further, overexpression of TRIM7 suppressed the replication of EMCV in HEK293T cells and enhanced the activity of IFN-ß promoter. On the other hand, knockdown of the endogenous TRIM7 promoted EMCV infection and impaired the activity of IFN-ß promoter. TRIM7 could regulate retinoic acid-inducible gene I (RIG-I)/ melanoma differentiation-associated gene 5 (MDA5)/ mitochondrial antiviral-signaling protein (MAVS) mediated IFN-ß signaling pathway. Moreover, TRIM7 interacted with MAVS and they were co-located in HEK293T cells. We demonstrate that TRIM7 plays a positive role in IFN-ß signaling pathway during EMCV infection and suppresses EMCV replication. Taken together, the presented results suggest that TRIM7 has a pivotal function in anti-EMCV infection, thereby providing a potential target for further development of anti-EMCV inhibitors.

A new B cell epitope of pC129R protein of African swine fever virus identified by monoclonal antibodies.

African swine fever virus (ASFV) is a most important pathogen which causes huge damage in swine production in the world. pC129R protein is one of the most abundant ASFV proteins in infected Vero cells and WSL-HP cells, which consequently could be a target for ASF detection and surveillance. In this study, 5-6-week-old female BALB/c mice were immunized with rpC129R protein expressed by a prokaryotic system. And three hybridomas, 1B1, 1B4 and 4H4, steadily secreted anti-pC129R monoclonal antibodies were screened by an indirect enzyme linked immunosorbent assay (ELISA). Among them, 1B4 and 4H4 had IgG2a isotype with Kappa light chain, while 1B1 had IgG1 isotype with Kappa light chain. Western blot and indirect immunofluorescence assay showed that three monoclonal antibodies (mAbs) specifically reacted with ASFV. Epitope mapping was performed with truncated polypeptides. And a new B cell epitope, 18KHYVLIPK25 was identified by the mAbs, which was highly conserved in most genotypes of ASFV. These findings not only provide a monoclonal antibody tool for further study of the function of C129R, but also lay the foundation for serological diagnosis and vaccine development.

The Establishment and Application of Indirect 3AB-ELISA for the Detection of Antibodies against Senecavirus A.

Senecavirus A (SVA) is an emerging pathogen that negatively affects the pig industry in China. Affected animals present vesicular lesions which are indistinguishable from other vesicular diseases. To date, there is no commercial vaccine that can be used to control SVA infection in China. In this study, recombinant SVA 3AB, 2C, 3C, 3D, L and VP1 proteins are expressed by using a prokaryotic expression system. The kinetics of the presence and levels of SVA antibodies with SVA-inoculated pig serum show that 3AB has the best antigenicity. An indirect enzyme-linked immunosorbent assay (ELISA) is developed with the 3AB protein, exhibiting a sensitivity of 91.3% and no cross-reaction with serum antibodies against PRRSV, CSFV, PRV, PCV2 or O-type FMDV. Given the high sensitivity and specificity of this approach, a nine-year (2014-2022) retrospective and prospective serological study is conducted to determine the epidemiological profile and dynamics of SVA in East China. Although SVA seropositivity declined markedly from 2016 (98.85%) to 2022 (62.40%), SVA transmission continues in China. Consequently, the SVA 3AB-based indirect ELISA has good sensitivity and specificity and is suitable for viral detection, field surveillance and epidemiological studies.

Porcine reproductive and respiratory syndrome virus infection triggers autophagy via ER stress-induced calcium signaling to facilitate virus replication.

Calcium (Ca2+), a ubiquitous second messenger, plays a crucial role in many cellular functions. Viruses often hijack Ca2+ signaling to facilitate viral processes such as entry, replication, assembly, and egress. Here, we report that infection by the swine arterivirus, porcine reproductive and respiratory syndrome virus (PRRSV), induces dysregulated Ca2+ homeostasis, subsequently activating calmodulin-dependent protein kinase-II (CaMKII) mediated autophagy, and thus fueling viral replication. Mechanically, PRRSV infection induces endoplasmic reticulum (ER) stress and forms a closed ER-plasma membrane (PM) contacts, resulting the opening of store operated calcium entry (SOCE) channel and causing the ER to take up extracellular Ca2+, which is then released into the cytoplasm by inositol trisphosphate receptor (IP3R) channel. Importantly, pharmacological inhibition of ER stress or CaMKII mediated autophagy blocks PRRSV replication. Notably, we show that PRRSV protein Nsp2 plays a dominant role in the PRRSV induced ER stress and autophagy, interacting with stromal interaction molecule 1 (STIM1) and the 78 kDa glucose-regulated protein 78 (GRP78). The interplay between PRRSV and cellular calcium signaling provides a novel potential approach to develop antivirals and therapeutics for the disease outbreaks.

African Swine Fever Virus Exhibits Distinct Replication Defects in Different Cell Types.

African swine fever virus (ASFV) causes one of the most devastating diseases affecting pigs and wild suids, a worldwide epizootic situation exacerbated in recent years due to the lack of vaccine or effective treatment. ASFV has a restricted cell tropism, and is prone to replicate in porcine monocytes and alveolar macrophages with high efficiency. Here, the replication capabilities of ASFV were examined in swine pulmonary alveolar macrophages (PAMs) and compared with 3D4/21, PK-15, MA-104 and Marc-145 cell lines using PCR, qPCR and Western blot with monoclonal antibodies against the viral p30 and p72 proteins. The results showed that ASFV has a variety of infection characteristics in PAMs and showed four cell lines with distinct defects during virus early transcription-translation, genome replication and late protein synthesis. Furthermore, an antiviral role of the stress granule pathway was revealed against ASFV, and ASFV infection inhibited stress granule formation in PAMs but not 3D4/21. These results will help to deepen our knowledge on ASFV infection and to develop ASFV susceptible cell lines.

Preparation of Monoclonal Antibodies against the Viral p54 Protein and a Blocking ELISA for Detection of the Antibody against African Swine Fever Virus.

African swine fever virus (ASFV) causes a highly contagious viral disease in domestic and wild pigs, leading to serious economic losses. As there are no vaccines or drugs available, early accurate diagnosis and eradiation of infected animals are the most important measures for ASFV prevention and control. Therefore, improvement of available diagnostic assays and development of novel effective techniques are required. This study is devoted to generating a new detection platform of blocking monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) against ASFV p54 protein. Seven monoclonal antibodies against recombinant p54 protein were produced and four epitopes were identified. Three blocking ELISAs were developed with 6A5 and 6F9 mAbs labeled with HRP, respectively, of which the 6A5/6F9-based blocking ELISA displayed the best detection performance, with an AUC of 0.986, sensitivity of 98.36% and specificity of 92.36% in ROC analysis. Moreover, it has an excellent agreement at 96.59% (198/205) when compared to the commercial blocking ELISA (kappa value = 0.920). The method also has high repeatability, with CV <10%, and no cross reaction with the serum antibodies against PRV, PRRSV, CSFV, PCV2 or SVA. This indicates that the 6A5/6F9-based blocking ELISA has high accuracy with good sensitivity and specificity, suitable for viral detection, field surveillance and epidemiological studies.

Vimentin promotes porcine circovirus type 2 (PCV2) replication in pig alveolar macrophage.

Porcine circovirus type 2 (PCV2) is a swine pathogen that causes postweaning multisystemic wasting syndrome, associated with a number of other disease symptoms in pigs. It has caused high economic losses for pig industries in China and worldwide. Vimentin is a cytoskeletal protein involved in the lifecycle of numerous viruses. In this study, the role of Vimentin in PCV2 replication in pig alveolar macrophage (3D4/21) cells was studied. A 3D4/21 cell line overexpressing Vimentin was established using a lentivirus expression system, in which PCV2 replication was significantly enhanced. Meanwhile, in the Vimentin silenced 3D4/21 cells, PCV2 replication was significantly reduced. Furthermore, we found that PCV2 infection in 3D4/21 cells promoted Vimentin phosphorylation and rearranged Vimentin distribution in cells. By inhibition of CaMKII pathway, Vimentin phosphorylation was significantly suppressed and PCV2 replication was significantly inhibited. The results in this study indicated that in 3D4/21 cells, PCV2 promoted CaMKIIγ-medicated Vimentin phosphorylation and altered its cellular distributions to accomplish its own replication.

Comparison of pathogenicity of different subgenotype porcine reproductive and respiratory syndrome viruses isolated in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen causing huge economic losses to the swine industry worldwide, because of its rapid evolution and variation in genomes. It is necessary to monitor the newly emerging epidemic strains and compare their virulence diversity in swine. In this study, two strains of PRRSV2 were isolated from clinical samples by using primary porcine alveolar macrophage (PAMs) cells and designated as SD1805 and FJ1805, respectively. They were unable to grow in MARC-145 cells. Full-length genomes sequence analysis revealed that SD1805 strain has the molecular characterization of NADC30 strain from American, and belongs to the branch of NADC30-like PRRSV (NL-PRRSV) (lineagea 1). FJ1805 isolate came from the inter-subgenotype recombinant of NADC30 strain with a highly pathogenic PRRSV (HP-PRRSV) strain HUN4. Moreover, it belongs to a lineagea 3 represented by the Chinese isolate QYYZ. Pathogenicity analysis showed that SD1805 strain had similar mortality and viral loads in lungs to HP-PRRSV BB0907 strain. FJ1805 strain showed milder pathogenicity compared to NL-PRRSV strain FJ1402 that was previously isolated with Marc-145 cells. It provides evidence of the circulation of the different subgenotype PRRSV strains in China with variations in cell adaption and pathogenic abilities.

A natural product, (S)-10-Hydroxycamptothecin inhibits pseudorabies virus proliferation through DNA damage dependent antiviral innate immunity.

Pseudorabies virus (PRV), a member of the subfamily alphaherpesvirinae, is one of the most important pathogenes that cause acute death in infected pigs and leads to substantial economic losses in the global swine industry. Recently, China's emerging PRV mutant strains resulted in the traditionally commercial vaccines not providing complete protection. Some studies reported that PRV could infect humans and cause endophthalmitis and encephalitis under certain circumstances. It is necessary to develop alternative manners to control the virus infection. Here, by screening a library of natural products, (S)-10-Hydroxycamptothecin (10-HCPT) was revealed to inhibit PRV replication with a selective index of 270.04. And 10-HCPT inhibited PRV replication by blocking the viral genome replication but not inhibiting the viral attachment, internalization, and release. RNA interference assay showed that 10-HCPT inhibited PRV replication by targeting DNA topoisomerase 1 (TOP1). Meanwhile, 10-HCPT treatment induced DNA damage response and stimulated antiviral innate immunity. Animal challenge experiments showed that 10-HCPT effectively alleviated clinical signs and hispathology, and increased INF-ß responses in lung and brain tissues of mice induced by PRV infection. The results demonstrate that 10-HCPT is a promising therapeutic agent to control PRV infection.