Co-infections involving human metapneumovirus (hMPV), respiratory syncytial virus (RSV), and influenza A virus (IAV) often exacerbate disease severity in vulnerable populations. Here, we employed a structure-based immunoinformatics approach to design a multi-epitope subunit vaccine targeting these pathogens. The construct incorporated two epitopes each for cytotoxic T lymphocytes (CTLs), helper T lymphocytes (HTLs), and B cells, derived from the fusion proteins of hMPV and RSV, as well as the neuraminidase protein of IAV. These epitopes were linked with an adjuvant and optimized spacers to enhance immunogenicity and structural stability. Structural modeling confirmed correct folding, and molecular docking predicted a stable interaction with Toll-Like Receptor 4 (TLR4) − 277.43 kcal/mol. Molecular dynamics simulations indicated a compact and stable complex with restricted conformational motions, while MM/GBSA analysis yielded a favorable binding free energy (–121.72 kcal/mol) dominated by electrostatic and van der Waals interactions. Immune simulations predicted strong humoral and cellular responses, including high antibody titers, IFN-γ and IL-2 production, and durable memory formation. Codon optimization achieved a codon adaptation index (CAI) of 0.98 and a GC content of 51.24%, suggesting efficient expression in Escherichia coli. These findings highlight the construct as a structurally stable, immunogenic, and expression-ready vaccine candidate, warranting experimental validation against hMPV, RSV, and IAV.

African swine fever (ASFV) leads a highly contagious and lethal hemorrhagic disease, causing a huge economic loss to the global pig industry. Recently, lethal genotype I/II recombinants were isolated and characterized in China and Vietnam. To differentiate the three genotypes (I, II and I/II recombinants) of ASFV in China, a triplex quantitative PCR (qPCR) assay was developed by targeting B646L, A151R and MGF360-14L genes. The detection limitation for the A151R, MGF360-14L, and B646L genes were 10 copies/μl, demonstrating a high sensitivity. Meanwhile, no cross-reactivity was observed with nucleic acids from ASFV genotype I and II or other swine viruses, confirming a high specificity for this assay. The coefficient of assay variation was below 2

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

Neuronal signals have emerged as pivotal regulators of B cells that regulate antitumor immunity and tumor progression. However, the functional relevance and mechanistic basis of the effects of the neurotransmitter dopamine (DA) on tumor immunity remain elusive. Here, we discovered that plasma DA levels are positively correlated with circulating B cell numbers and potently activate B cell responses in a manner dependent on the DRD5 receptor. Notably, DRD5 signaling enhanced the Janus kinase 1 (JAK1)-STAT1 signaling in B cell responses, which enhanced B cell activation and increased antigen presentation and co-stimulation, resulting in increased expansion and cytotoxicity in tumor-specific effector of T cells. Our findings demonstrate that DA signaling suppresses tumor progression and highlight DRD5 as a promising target for cancer immunotherapy.

ABSTRACT Hepatitis E virus (HEV) is a viral hepatitis pathogen that poses a significant threat to global human health, representing a serious yet long-overlooked public health concern. In this study, we identified glucose-regulated protein 75 (GRP75) as an interaction partner of HEV-ORF2 using recombinant ORF2 truncation as bait. The substrate-binding domain of GRP75 interacted with HEV-ORF2 and inhibited HEV replication by facilitating HEV-ORF2 degradation. Further analysis revealed that HEV-ORF2 contains three KFERQ-like motifs, the key signature sequence required for chaperone-mediated autophagy (CMA). Our data demonstrated that GRP75-mediated degradation of HEV-ORF2 was heat-shock cognate protein 70 (HSC70)-dependent, although no direct interaction between HSC70 and ORF2 was detected. Instead, GRP75, together with HEV-ORF2 and HSC70, formed a complex that mediated CMA-dependent degradation of HEV-ORF2, whereas deletion of all three KFERQ-like motifs from ORF2 conferred resistance to such processes. Additionally, GRP75 blocked mitochondrial transport of HEV-ORF2, potentially mitigating ORF2’s function as an interferon (IFN) induction antagonist. Furthermore, GRP75 enhanced the interaction between mitochondrial antiviral signaling protein (MAVS) and TANK-binding kinase 1 (TBK1), promoting IFN-β production and ultimately inhibiting HEV infection. In conclusion, our findings identify GRP75 as a novel restriction factor for HEV infection and provide new insights into its role in CMA and antiviral innate immunity.IMPORTANCEDue to the lack of an effective in vitro model, the viral-host interaction of HEV remains largely elusive. This study uncovers a novel mechanism by which GRP75 inhibits HEV infection. On one hand, the GRP75 protein facilitates the degradation of HEV-ORF2 through the lysosome-associated, chaperone-mediated autophagy by recognizing KFERQ-like motif presented on HEV-ORF2. On the other hand, GRP75 enhances the production of IFN-β by promoting interaction between MAVS and TBK1, thereby establishing an antiviral state and suppressing HEV infection. This research expands our current understanding of host resistance to HEV and provides a new function of GRP75, suggesting that GRP75 might be a novel antiviral factor against virus infection.

IFN-λs hold promise as therapeutic candidates against mutable respiratory viruses, but their efficacy against porcine reproductive and respiratory syndrome virus (PRRSV) remains unclear. In this study, we expressed a recombinant fusion protein consisting of porcine ISG15 linked porcine IFN-λ3 (ISG15-IFN-λ3) via a rigid protein linker in Escherichia coli (E. coli). In vitro experiments demonstrated that treatment of porcine alveolar macrophage (PAM)-derived CRL-2843 cells with ISG15-IFN-λ3 induced upregulation of several Interferon-stimulated Genes (ISGs) proteins, including ISG15, ISG56, and HERC5. CRL-2843 cells pretreated with ISG15-IFN-λ3 exhibited heightened resistance to Newcastle disease virus infection, while PRRSV-permissive cells treated with ISG15-IFN-λ3 before and during PRRSV exposure showed significantly inhibited PRRSV replication as well. In animal experiments, at 21 days post-infection, ISG15-IFN-λ3-treated piglets displayed milder lung tissue pathology and significantly reduced serum PRRSV-RNA, indicating enhanced viral clearance and faster recovery. Additionally, PAMs collected from ISG15-IFN-λ3-treated piglets showed significantly reduced mRNA expression of representative cytokines, chemokines, suggesting that ISG15-IFN-λ3 treatment may mitigate pneumonia severity by reducing the levels of these inflammatory mediators. These findings indicate that recombinant ISG15-IFN-λ3 expressed in E. coli may serve as a novel, effective, and affordable agent for treating severe PRRSV infection in piglets, potentially benefiting the pork industry.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a significant threat to the global pork industry, resulting in substantial economic losses. Current control measures rely on modified live virus (MLV) vaccines with safety concerns. However, the lack of consensus on protective PRRSV antigens is impeding the development of effective and safety subunit vaccines. In this study, we conducted in vitro virus neutralization (VN) assays in MARC-145 and CRL-2843CD163/CD169 cell lines and primary porcine alveolar macrophages (PAMs) to systemically identify PRRSV structural proteins (SPs) recognized by virus-neutralizing antibodies in hyperimmune serum collected from piglets infected with highly pathogenic PRRSV (HP-PRRSV). Additionally, piglets immunized with different combinations of recombinant PRRSV-SPs were challenged with HP-PRRSV to evaluate their in vivo protection potential. Intriguingly, different in vitro VN activities of serum antibodies elicited by each PRRSV SP were observed depending on the cell type used in the VN assay. Notably, antibodies specific for GP3, GP4, and M exhibited highest in vitro VN activities in PAMs, correlating with complete protection (100% survival) against HP-PRRSV challenge in vivo after immunization of piglets with combination of GP3, GP4, M and N (GP3/GP4/M/N). Further analysis of lung pathology, weight gain, and viremia post-challenge revealed that the combination of GP3/GP4/M/N provided superior protective efficacy against severe infection. These findings underscore the potential of this SP combination to serve as an effective PRRSV subunit vaccine, marking a significant advancement in pork industry disease management.

Interferon-stimulated gene product 15 (ISG15) can be conjugated to substrates through ISGylation. Currently, the E3 ligase for porcine ISGylation remains unclear. Here, we identified porcine HERC5 and HERC6 (pHERC5/6) as ISGylation E3 ligases with pHERC6 acting as a major one by reconstitution of porcine ISGylation system in HEK293 T cell via co-transfecting E1, E2 and porcine ISG15(pISG15) genes. Meanwhile, our data demonstrated that co-transfection of pISG15 and pHERC5/6 was sufficient to confer ISGylation, suggesting E1 and E2 of ISGylation are interchangeable between human and porcine. Using an immunoprecipitation based ISGylation analysis, our data revealed pHERC6 was a substrate for ISGylation and confirmed that K707 and K993 of pHERC6 were autoISGylation sites. Mutation of these sites reduced pHERC6 half-life and inhibited ISGylation, suggesting that autoISGylation of pHERC6 was required for effective ISGylation. Conversely, sustained ISGylation induced by overexpression of pISG15 and pHERC6 could be inhibited by a well-defined porcine ISGylation antagonist, the ovarian tumor (OTU) protease domain of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-nsp2 and PRRSV-nsp1 beta, further indicating such method could be used for identification of virus-encoded ISG15 antagonist. In conclusion, our study contributes new insights towards porcine ISGylation system and provides a novel tool for screening viral-encoded ISG15 antagonist.

Hepatitis E virus (HEV) is a common causative agent of acute hepatitis. Due to the shortage of efficient in vitro model, the viral assembly and release processes are still poorly understood. In this study, we found that ORF3, an HEV structural protein showed prion-like properties. The prion domain (PrD) of yeast prion Sup35 could be functionally replaced by HEV-ORF3 and its candidate PrD (cPrD). A single amino acid substitution in the cPrD reduced the aggregation propensity of ORF3 and blocked the function of ORF3 in enhancing the stability of microtubules in HEV-infected cells, thus blocking virion release from the infected cells and resulting in reduced HEV pathogenicity in Mongolian gerbils. These data suggest that HEV-ORF3 is a novel functional prion-like protein which assembles into a filamentous structure for virion release, supporting the hypothesis that the self-propagating properties of prion proteins are widely exploited as epigenetic information carriers in nature.

Granulocyte-macrophage colony stimulating factor (GM-CSF), participates in diverse biological processes associated with innate and adaptive immunity, has unknown effects during PRRSV infection. Here, a double-antibody sandwich ELISA for pGM-CSF was developed in-house for evaluation of pGM-CSF level during PRRSV infection both in vitro and in vivo. In in vitro assay, it was notable that PRRSV-infected porcine alveolar macrophages (PAMs) yielded inconsistent pGM-CSF protein- and mRNA-level, suggesting a post-transcriptional inhibition of pGM-CSF mRNA was employed by PRRSV. Meanwhile, concurrent analysis of pGM-CSF levels in serum samples from PRRSV-infected piglets suggested that effect of PRRSV infection demonstrated minimum effect on pGM-CSF levels regardless of PRRSV virulence phenotypes. Moreover, in vitro treatment of PAMs with pGM-CSF prior PRRSV inoculation did not inhibit PRRSV replication in PAMs although genes downstream of pGM-CSF in PAMs could be upregulated by pGM-CSF treatment. Meanwhile, knockdown of pGM-CSF using siRNA did not enhance PRRSV replication as well. Intriguingly, therapeutic antibody treatment of HP-PRRSV-infected piglets led to significantly increased serum pGM-CSF levels, thus aligning with low pneumonia incidence and low intracellular PRRSV-RNA levels in PAMs of therapeutic antibody treated piglets. Furthermore, transcriptome analysis of PAMs from infected piglets revealed increased serum pGM-CSF levels correlated with activation of downstream signal of pGM-CSF in PAMs as evidenced by a M1-like phenotypes of gene expression pattern, implying a potential host-protective role played by pGM-CSF for PRRSV infection in vivo. In conclusion, our results demonstrated developments of a highly sensitive and specific ELISA for pGM-CSF and revealed a potential protective role conferred by pGM-CSF during PRRSV infection.

Current strategies for porcine reproductive and respiratory syndrome (PRRS) control are inadequate and mainly restricted to immunization using different PRRS virus (PPRSV) vaccines. Although there are no safety concerns, the poor performance of inactivated PRRSV vaccines has restricted their practical application. In this research, we employed the novel PRRSV-specific IgM monoclonal antibody (Mab)-PR5nf1 as a vaccine adjuvant for the formulation of a cocktail composed of inactivated PRRSV (KIV) and Mab-PR5nf1 along with a normal adjuvant to enhance PRRSV-KIV vaccine-mediated protection and further compared it with a normal KIV vaccine and modified live virus vaccine (MLV). After challenge with highly pathogenic (HP)-PRRSV, our results suggested that the overall survival rate (OSR) and cell-mediated immunity (CMI), as determined by serum IFN-γ quantification and IFN-γ ELISpot assay, were significantly improved by adding PRRSV-specific IgM to the PRRSV-KIV vaccine. It was also notable that both the OSR and CMI in the Mab-PR5nf1-adjuvanted KIV group were even higher than those in the MLV group, whereas the CMI response is normally poorly evoked by KIV vaccines or subunit vaccines. Compared with those in piglets immunized with the normal KIV vaccine, viral shedding and serum neutralizing antibody levels were also improved, and reduced viral shedding appeared to be a result of enhanced CMI caused by the inclusion of IgM as an adjuvant. In conclusion, our data provide not only a new formula for the development of an effective PRRSV-KIV vaccine for practical use but also a novel method for improving antigen-specific CMI induction by inactivated vaccines and subunit vaccines.

Porcine epidemic diarrhea (PED) is an acute, highly contagious intestinal swine disease caused by porcine epidemic diarrhea virus (PEDV). In addition to known PEDV infection targets (villous small intestinal epithelial cells), recent reports suggest that dendritic cells (DCs) may also be targeted by PEDV in vivo. Thus, in this study we used bone marrow-derived dendritic cells (BM-DCs) as an in vitro model of antigen-presenting cells (APCs). Our results revealed that PEDV replicated in BM-DCs and that PEDV infection of cells inhibited expression of swine leukocyte antigen II DR (SLA-DR), a key MHC-II molecule involved in antigen presentation and initiation of CD4+ T cell activation. Notably, SLA-DR inhibition in BM-DCs did not require PEDV replication, suggesting that PEDV structural proteins participated in SLA-DR transcriptional inhibition. Moreover, reporter assay-based screening indicated that PEDV envelope protein blocked activation of SLA-DRα and β promoters, as did PEDV-ORF3 protein when present during PEDV replication. Meanwhile, treatment of PEDV-infected BM-DCs with MG132, a ubiquitin-proteasome degradation pathway inhibitor, did not restore SLA-DR protein levels. Additionally, PEDV infection of BM-DCs did not alter SLA-DR ubiquitination status, suggesting that PEDV infection did not affect SLA-DR degradation. Furthermore, additions of PEDV structural proteins to HEK-293T-SLA-DR stably transfected cells had no effect on SLA-DR protein levels, indicating that PEDV-mediated inhibition of SLA-DR expression acted mainly at the transcriptional level, not at the protein level. These results provide novel insights into PEDV pathogenic mechanisms and viral-host interactions.

Interferon-stimulated gene product 15 (ISG15), a ubiquitin-like molecule, can be conjugated to protein substrates through a reversible process known as ISGylation. ISG15 and ISGylation are both strongly upregulated by type I interferons and play putative key roles in host innate immunity against viral infection. However, the function of ISGylation and identities of ISGylation substrates are largely unknown. Here, a novel monoclonal antibody (Mab) that specifically recognizes porcine ISG15 (pISG15) was employed to capture ISG15-conjugated proteins from IFNs-stimulated porcine cell lysates. Next, Mab-captured conjugates were analyzed using proteomics-based tools to identify potential ISGylation protein targets in order to elucidate the roles of ISG15 and ISGylation in porcine cells. Subsequently, 190 putative ISGylation sites were detected within 98 identified ISGylation candidates; several candidates contained more than one ISGylation-modifiable lysine residue, including pISG15 itself. Motif enrichment analysis of confirmed ISGylation sites demonstrated a moderate bias towards certain sites with specific upstream amino acid residues. Meanwhile, results of Gene Ontology (GO)-based annotation and functional enrichment and protein-protein interaction (PPI) network analyses of porcine ISG15-conjugated substrate proteins indicated that these substrates were mainly associated with the host metabolism, especially nucleotide metabolic pathways that ultimately may participate in cellular antiviral defenses. Notably, several ISGs (MX1, IFIT1, OAS1, ISG15 and putative ISG15 E3 ligase Herc6) were also identified as putative ISGylation substrates within a regulatory loop involving ISGylation of ISGs themselves. Taken together, proteomics analysis of porcine ISGylation substrates revealed putative functional roles of ISG15 and novel host ISGylation targets that may ultimately be involved in cellular antiviral responses.

Hepatitis E virus (HEV) is a zoonotic pathogen causing hepatitis in both human and animal hosts, which is responsible for acute hepatitis E outbreaks worldwide. The 7.2 kb genome of the HEV encodes three well-defined open reading frames (ORFs), where the ORF2 translation product acts as the major virion component to form the viral capsid. In recent years, besides forming the capsid, more functions have been revealed for the HEV-ORF2 protein, and it appears that HEV-ORF2 plays multiple functions in both viral replication and pathogenesis. In this review, we systematically summarize the recent research advances regarding the function of the HEV-ORF2 protein such as application in the development of a vaccine, regulation of the innate immune response and cellular signaling, involvement in host tropism and participation in HEV pathogenesis as a novel secretory factor. Progress in understanding more of the function of HEV-ORF2 protein beyond the capsid protein would contribute to improved control and treatment of HEV infection.

Neutralizing antibodies (NAbs) have attracted attention as tools for achieving PRRSV control and prevention, but viral antigenic variation undermines the abilities of NAbs elicited by attenuated PRRSV vaccines to confer full protection against heterogeneous PRRSV field isolates. As demonstrated in this study, the monoclonal antibody (mAb) mAb-PN9cx3 exhibited broad-spectrum recognition and neutralizing activities against PRRSV-1 and PRRSV-2 strains in vitro. Furthermore, in vivo experiments revealed that the administration of two 10-mg doses of mAb-PN9cx3 before and after the inoculation of piglets with heterologous PRRSV isolates (HP-PRRSV-JXA1 or PRRSV NADC30-like strain HNhx) resulted in significant reduction of the PRRSV-induced pulmonary pathological changes and virus loads in porcine alveolar macrophages (PAMs) compared with the results obtained with mAb-treated isotype controls. Moreover, minimal hilar lymph node PRRSV antigen levels were observed in mAb-PN9cx3-treated piglets. A transcriptome profile analysis of PAMs extracted from lung tissues of piglets belonging to different groups (except for antibody-isotype controls) indicated that mAb-PN9cx3 treatment reversed the PRRSV infection-induced alterations in expression profiles. A gene ontology (GO) enrichment analysis of these genes traced their functions to pathways that included the immune response, inflammatory response, and response to steroid hormone, and their functions in oogenesis and positive regulation of angiogenesis have been implicated in PRRSV pathogenesis. Overall, NADC30-like HNhx infection affected more gene pathways than HP-PRRSV infection. In conclusion, our research describes a novel immunologic approach involving the use of mAbs that confer cross-protection against serious illness resulting from infection with heterogeneous PRRSV-2 isolates, which is a feat that has not yet been achieved through vaccination. Ultimately, mAb-PN9cx3 will be a powerful addition to our current arsenal for achieving PRRSV prevention and eradication.

African swine fever virus (ASFV), one of the most devastating emerging swine pathogens in China, causes nearly 100 % mortality in naive herds. Here, whole-transcriptome RNA-seq analysis was conducted in porcine alveolar macrophages (PAMs) infected with Pig/Heilongjiang/2018 (Pig/HLJ/18) ASFV at different time points. Our data suggested that ASFV genes expression demonstrated a time-depended pattern and ASFV early genes were involved in antagonizing host innate immunity. Moreover, viral small RNA (vsRNA) was generated as well. Meanwhile, transcriptome analysis of host genes suggested a strong inhibition host immunity-related genes by ASFV infection in PAMs, while enhanced chemokine-mediated signaling pathways and neutrophil chemotaxis were observed in ASFV infected PAMs. Furthermore, ASFV infection also down-regulated host microRNAs (miRNAs) that putatively targeted viral genes, while also triggering dysregulation of host metabolism that promoted virus replication at transcription level. Most importantly, infection of PAMs with ASFV induced a different transcriptome pattern from that of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), which is known to trigger a host cytokine storm. In conclusion, our transcriptome data implied that ASFV infection in PAMs appeared to be associated with strong inhibition of host immune responses, dysregulation of host chemokine axis and metabolic pathways.

PRRSV has haunted the swine industry for over 30 years since its emergence. Besides the limited efficacy of PRRSV modified live vaccines (MLVs) against heterogeneous PRRSV isolates, rapid induction of nonneutralizing antibodies (non-NAbs) against PRRSV NSPs after MLV immunization or wild-strain infection is one of the reasons why development of an effective vaccine has been hampered. By using in vitro -generated BMDCs as models to understand the antigen presentation process of PRRSV, we obtained data indicating that PRRSV infection of BMDCs promotes functional SLA-DR upregulation to present PRRSV NSP-derived immunopeptides for evoking a non-NAb response in vivo . Our work not only uncovered a novel mechanism for interference in host antigen presentation by PRRSV but also revealed a novel insight for understanding the rapid production of nonneutralizing antibodies against PRRSV NSPs, which may have benefit for developing an effective vaccine against PRRSV in the future.

Although widespread administration of attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccines has been implemented since they first became commercially available two decades ago, PRRSV infection prevalence in swine herds remains high. The limited success of PRRSV vaccines is partly due to the well-established fact that a given vaccine strain confers only partial or no protection against heterologous strains. In our past work, A2MC2-P90, a novel PRRSV vaccine candidate that induced a type I IFNs response in vitro, conferred complete protection against challenge with genetically heterologous PRRSV strains. Here we assessed the ability of the PRRSV vaccine candidate A2MC2-P90 to protect piglets against the HP-PRRSV challenge and compared its efficacy to that of a licensed HP-PRRSV-specific vaccine (TJM-F92) assessed in parallel. A2MC2-P90 provided vaccinated piglets with 100% protection from a lethal challenge with extremely virulent HP-PRRSV-XJA1, while 100% mortality was observed for unvaccinated piglets by day 21 post-challenge. Notably, comparison of partial sequence (GP5) of XJA1 to A2MC2-P90 suggested there was only 88.7% homology. When comparing post-HP-PRRSV challenge responses between piglets administered A2AMC2-P90 versus those immunized with licensed vaccine TJM-F92, A2MC2-P90-vaccinated piglets rapidly developed a stronger protective humoral immune response, as evidenced by much higher titers of neutralizing antibodies, more rapid clearance of viremia and less nasal virus shedding. In conclusion, our data suggest that this novel vaccine candidate A2MC2-P90 has improved protection spectrum against heterologous HP-PRRSV strains.

Porcine reproductive and respiratory syndrome (PRRS) is the most economically important infectious disease affecting the global swine industry, especially since vaccination has had limited impact on PRRSV prevention and control. In this study, the monoclonal antibody PR5nf1 (Mab-PR5nf1, IgM isotype) was shown to react with heterogeneous PRRSV isolates belonging to both PRRSV-1 and PRRSV-2 species. Pepsin digestion of Mab-PR5nf1 did not affect Mab binding to virions, as F(ab)2 fragments demonstrated the same reactivity as undigested Mab. Upon further investigation, Mab-PR5nf1 could neutralize all tested PRRSV isolates of both PRRSV-1 and PRRSV-2, suggesting it was a broadly neutralizing Mab against PRRSV. Interestingly, Mab-PR5nf1 appeared to recognize a specific virus epitope that required post-translational modification within the host cellular Golgi apparatus. Deglycosylation of PRRSV virions with PNGase F abolished Mab binding, suggesting that a novel Mab-binding epitope may exist that confers cross-protection against isolates of both PRRSV species. Additionally, immunization of mice with a cocktail of inactivated PRRSV virus and Mab-PR5nf1 enhanced cell-mediated immunity, as determined by IFN-γ ELIspot. In conclusion, this is the first report describing a novel Mab that recognizes a conserved epitope common to both PRRSV-1 and PRRSV-2 and provides valuable insights to guide future PRRSV vaccine development.