Currently, monkeypox virus (MPXV) treatment methods face a dilemma of potential drug resistance. They cannot simultaneously address two core pathological mechanisms driving infection: continuous viral replication and uncontrolled inflammatory storm. To overcome these challenges, we innovatively developed a dual-function therapeutic strategy with precise virus clearance and anti-inflammatory effects, constructing a dual hydrogel system synergistically combining phototherapy and immunomodulatory effects. Specifically, aggregation-induced emission (AIE) nanoparticles (NPs) are embedded in a reactive oxygen species (ROS)-degradable polyvinyl alcohol (PVA)-N1-(4-borobenzoyl)-N3-(4-borobenzoyl)-the N1, the N1, N3, N3-tetramethylpropane-1,3-diamine (tsPBA) hydrogel, while the anti-inflammatory agent is loaded in a zwitterionic hydrogel (SBMA) matrix. Under near-infrared band laser irradiation, AIE NPs efficiently generate large amounts of ROS, which, together with inflammation-produced endogenous ROS, trigger the rapid degradation of the ROS-responsive hydrogel and release of AIE NPs. The generated ROS effectively destroys the MPXV shell and strongly inactivates the virus. After virus clearance, the SBMA hydrogel continuously releases anti-inflammatory drugs (Dexamethasone, DXMS) to inhibit excessive pro-inflammatory cytokines and reduce inflammation-induced tissue damage. In mouse, rabbit, and non-human primate models with MPXV-induced skin damage, this hydrogel nanoparticle protective layer significantly inhibits viral replication and accelerates wound healing, representing a transformative, convertible platform for treating MPXV and other highly inflammatory viral infections.

Getah virus (GETV), a mosquito-borne alphavirus, poses an emerging threat to public health with its increasingly broad host spectrum. While glycosaminoglycans (GAGs) serve as critical attachment factors for many alphaviruses and the low-density lipoprotein receptor (LDLR) facilitates the cellular entry of several members, the precise viral determinants governing these interactions and their implications for viral virulence remain poorly defined. Here, we introduced an H86Y substitution, a potential adaptive mutation site, within the E2 glycoprotein of GETV using reverse genetics. The H86Y mutant replicated more efficiently in mosquito C6/36 cells but was consistently attenuated across several mammalian cell lines. In susceptible mouse models, H86Y infection led to reduced viral loads, milder histopathology, and lower inflammatory responses compared with the parental virus, yet still elicited robust protective immunity in adult mice. Mechanistically, a series of functional assays, including infection in GAG-deficient cells, decoy inhibition, co-immunoprecipitation, receptor overexpression and knockdown, and biolayer interferometry, demonstrated that the residue 86 in E2 glycoprotein is a critical determinant for GETV binding to both GAGs and LDLR. The H86Y mutation concurrently reduces these interactions, contributing the impairment of virus attachment and entry into mammalian cells. Furthermore, the GAG-binding site functionally overlaps with the LDLR interaction interface. In LDLR-deficient suckling mice, the impaired replication of H86Y persisted in examined tissues. However, pre-treatment with heparinase nearly completely eliminated this attenuation phenotype, further confirming that LDLR and GAG are the key host factors mediating attenuation phenotype for H86Y. In summary, the residue 86 of the GETV E2 glycoprotein represents a determinant of viral virulence, and an H86Y mutation attenuates GAGs and LDLR-dependent infection, providing mechanistic insights into alphavirus-host interactions and a potential target for antiviral and vaccine development.

Orf virus (ORFV) is a member of the Parapoxvirus genus of the Poxviridae family causing contagious diseases in sheep, goats, and wild ungulates, with zoonotic potential in humans. Although many viruses, including poxviruses, are known to utilize the host cellular machinery to reproduce viral particles, the metabolic changes induced by ORFV remain unclear. In the present study, non-targeted metabolomics and proteomics were employed to investigate the impact of ORFV infection on the host cellular metabolism network. A total of 301 metabolites and 802 proteins were significantly altered during the early stages of ORFV infection, and most of them were involved in cellular lipid metabolism, amino acid metabolism, nucleotide metabolism, and glucose metabolism. We further determined the effect of the host's metabolic system on ORFV replication using the TCID50 assay. Virus titers were significantly decreased in the absence of glucose or when treated with the de novo fatty acid synthesis inhibitor, indicating that glucose metabolism and de novo fatty acid synthesis pathway were required for ORFV replication. However, glutamine did not affect viral titers. Our findings provide insights into ORFV-host interactions, which are critical for developing new preventive or therapeutic strategies against ORFV by targeting altered metabolic pathways.

The STING pathway is pivotal in defense against RNA viruses; however, its involvement in swine acute diarrhea syndrome coronavirus (SADS-CoV) infection remains unclear. This study reveals a dual mechanism where in SADS-CoV triggers STING activation via nuclear envelope rupture but subsequently evades immunity through its nucleocapsid (N) protein. Mechanistically, this process involves chromatin leakage that activates the cGAS-STING pathway, triggering interferon responses. The endoplasmic reticulum-resident protein defender against cell death 1 (DAD1) plays a key role in promoting STING phosphorylation and trafficking to the Golgi apparatus. The SADS-CoV N protein binds to STING to block its activation and translocation, disrupting DAD1-mediated antiviral signaling. Notably, the E368A mutation in the N protein weakens STING binding and impairs immune suppression. These findings reveal that the SADS-CoV N protein evades host innate immunity by disrupting DAD1-mediated activation, pointing to potential targets for antiviral strategies.

Toll-like receptors (TLRs) play a pivotal role in the innate immune system by recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), thereby initiating immune responses against viral infections. TLR agonists have emerged as promising adjuvants to enhance the efficacy of viral vaccines by modulating immune responses, improving antigen presentation, and promoting both humoral and cellular immunity. This review comprehensively summarizes the classification, signaling mechanisms, and immunomodulatory functions of cell-surface and intracellular TLRs. It further discusses the application of TLR agonists as adjuvants in vaccines against major viruses, including HBV, HCV, HIV, SARS-CoV-2, influenza, and flaviviruses. Key findings from preclinical and clinical studies highlight the potential of TLR agonists to overcome immune tolerance, enhance vaccine immunogenicity, and provide broad-spectrum protection. Finally, it points toward the “integration of precision adjuvants with novel vaccine platforms” as a core future direction, laying a theoretical and applied foundation for TLR agonists to become the next generation of viral vaccine adjuvants.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, remains a leading cause of viral encephalitis in Asia and poses serious threats to reproductive health. However, the molecular mechanisms underlying JEV-induced testicular inflammation remain incompletely understood. In this study, an integrated approach involving proteomic, cellular, and animal experiments was used to investigate the role of Toll-like receptor 4 (TLR4) in JEV-triggered inflammatory responses. Proteomic analysis and in vitro assays revealed that JEV infection upregulated TLR4 expression and activated the NF‑κB signaling cascade, resulting in enhanced secretion of inflammatory cytokines, including TNF-α, IL-1β, and IL-6. Among the viral proteins examined, only the envelope (E) protein significantly increased endogenous TLR4 levels in a dose-dependent manner. Direct interaction between the E protein and TLR4 was confirmed by co-immunoprecipitation and immunofluorescence. In a mouse model of JEV infection, the virus induced severe testicular inflammation, characterized by tissue disruption, TLR4 upregulation, and activation of the TLR4/NF-κB pathway, consistent with the in vitro observations. Collectively, these findings identify the JEV E protein as a key inducer of TLR4-mediated inflammation and provide mechanistic insights into JEV-associated testicular pathology.

Oncolytic viruses (OVs) can selectively replicate in tumor cells and trigger anti-tumor immunity. However, it remains unclear whether OVs can target the process of ‘information transmission’ between tumor cells to assist their anti-tumor effects. Uncovering this issue would help to understand the way how OV reshapes tumor microenvironment. Here, we focused on the interaction between oncolytic parapoxvirus (ORFV) and tumor-derived extracellular vesicles (EVs), attempting to reveal the regulatory role of OVs in the process of tumor information transmission. We previously constructed a recombinant orf virus ORFV△ with virulence gene deletion as an anti-tumor agent. We found that tumor-derived EVs could interfere with the anti-tumor growth and metastasis capabilities of ORFV△. Moreover, we also found that ORFV△ inhibited the level of tumor-derived EVs, indicating that ORFV△ and EVs can affect with each other. Notably, ORFV△ can further reprogram microRNA (miRNA) profile in EVs, prompting EVs to enrich a series of miRNAs that target oncogenes in pro-tumor pathways. Subsequently, it was confirmed that the EVs produced by tumors treated with ORFV△ have significantly restricted abilities in promoting tumor cell viability, proliferation, and migration. The above results enrich the anti-tumor mechanism of OV and suggest the necessity of inhibiting tumor-derived EVs during OV-based therapy.

Acinetobacter baumannii (A. baumannii), a very common pathogen, poses a significant public health threat due to its antibiotic resistance and long survival in healthcare environments. Both A. baumannii and carbapenem-resistant A. baumannii (CRAB) can spread through the air, increasing infection risks. Therefore, monitoring their presence in the air is of great significance, especially in hospitals. Herein, we developed a Chelex-100-LAMP-CRISPR/Cas12a (CLC) platform including DNA release and nucleic acid test. Combined with a wet cyclone sampler, the platform can detect airborne A. baumannii and its most common carbapenem-resistant gene, blaOXA-23, within 70 min. This CLC platform has also been proven to have a detection limit of 6 × 102 CFU of CRAB per test through simulated air samples. Moreover, this platform was also used to test five actual air samples from a tertiary hospital, and the results achieved perfect concordance with sequencing data, validating the platform’s accuracy and reliability. Therefore, the CLC platform showed great potential for the rapid, on-site detection of airborne A. baumannii and its carbapenem-resistant gene blaOXA-23, offering a valuable tool for infection control in healthcare environments.

The recent global outbreaks of mpox highlight the urgent need for both fundamental research and antiviral development. However, studying the mpox virus (MPXV), with its large and complex genome, remains challenging due to the requirement for high-containment facilities. Here, we describe a strategy for de novo assembly of MPXV clade IIb genomes in bacterial artificial chromosomes using transformation-associated recombination cloning. Leveraging CRISPR-Cas9 and Lambda Red recombination, we engineer replication-defective MPXV particles with dual deletions of OPG96 (M2R) and OPG158 (A32.5 L)-genes essential for virion assembly, that are capable of recapitulating key stages of the viral life cycle. We apply this system to screen a compound library and identify G243-1720, a potent anti-poxvirus inhibitor with broad activity in vitro and in vivo. G243-1720 blocks the formation of extracellular enveloped virions and cell-cell spread. Resistance mutation selection, crystallographic analysis, analytical ultracentrifugation, and mass photometry reveal that, despite its distinct chemical structure, G243-1720 shares a mode of action with tecovirimat, both functioning by affecting dimerization of protein OPG57 (F13). Our findings underscore the potential of G243-1720 as a promising broad-spectrum anti-poxvirus lead compound and demonstrate the utility of replication-defective MPXV particles as a reliable platform for viral biology studies and antiviral development.

Currently, mpox virus (MPXV) continues to pose a global public health challenge, with immunocompromised individuals often exhibiting more severe clinical symptoms. This study screens three male mouse models (ICR, IFNAR1-/-, SCID) and identifies SCID mice as the optimal model for modeling severe patient symptoms, including pneumonia, rash, and localized inflammation, which is applied to evaluate the antiviral efficacy of tecovirimat and cidofovir. Both drugs prevent systemic MPXV spread when administered within two days post virus exposure. Local antiviral efficacy differs between the two drugs, particularly after intradermal infection. Prolonged treatment up to 28 days post infection results in 100% survival of SCID mice but fails to effectively suppress localized rash and inflammatory swelling, suggesting that the drugs have limited impact at later stages of the disease. These findings indicate that the therapeutic efficacy of tecovirimat and cidofovir depends on the timing of intervention initiation and the stage of disease progression.

Getah virus (GETV) is a mosquito-borne alphavirus. It has posed a threat to various livestock and humans during decades of evolution and spread. However, the viral determinants and mechanisms underlying enhanced virulence remain largely unknown. In this study, we identified E2-N262 glycosylation in GETV as an evolutionary adaptation that promotes receptor binding, innate immune evasion, and virulence. Using reverse genetics technology, we generated recombinant GETV strains-rSD2206 WT and the N262D substitution. In vitro, rSD2206 WT formed larger plaques and replicated more efficiently across multiple cell lines. Despite exhibiting enhanced pathogenicity, higher replication efficiency, and a weaker induction of host antiviral responses compared to the N262D virus in both mouse and suckling pig models, the rSD2206 WT virus retained immunogenicity. Mechanistically, we found that rSD2206 WT increased virion attachment and entry into host cells by enhancing binding affinity to both HS (a GETV attachment factor) and LDLR (a GETV receptor). In addition, the rSD2206 WT strain can evade the host's antiviral response by interrupting the activation of interferon regulatory factor 3. Structural analysis revealed a significant reduction in binding free energy for rSD2206 WT. This reduction resulted in conformational changes within the E2 protein, which in turn enhanced its stability and subsequently strengthening its receptor-binding capacity. Ultimately, compared to N262D, rSD2206 WT exhibited increased replication in cells overexpressing LDLR, but significantly reduced replication in LDLR-/- mice. These results highlight the critical importance of proactive surveillance of GETV molecular evolution to prevent future outbreaks, which could provide the foundation for the development of attenuated vaccines.

The recently identified alphacoronavirus swine acute diarrhea syndrome coronavirus (SADS-CoV) has a high fatality rate in neonatal piglets. Currently, no vaccines or treatment strategies for SADS-CoV infection are available. The stimulator of interferon genes (STING) pathway plays a critical role in initiating innate immune responses against RNA viral infections; however, its role in host defense against SADS-CoV infection remains unexplored. We assessed the pathogenicity of SADS-CoV in 3-day-old, 7-day-old, and 3-week-old mice, revealing striking age-dependent susceptibility-a pattern mirroring clinical observations in piglets. Additionally, SADS-CoV infection activated the STING-dependent pathway, which resulted in significant interferon responses in infected mice. In vitro experimental findings confirmed that STING pathway activation inhibited SADS-CoV replication by modulating the NF-κB and IRF3 signaling pathways and mediating the production of inflammatory cytokines, which underscores the importance of the STING pathway in antiviral defense mechanisms. In vivo studies revealed that the STING inhibitor C176 significantly promoted viral replication, whereas activation of the STING pathway using the STING agonist diABZI increased antiviral immune responses and reduced viral replication. Notably, diABZI protected mice from SADS-CoV infection by reducing viral replication through mechanisms involving both type I interferon-dependent and -independent pathways. These results represent the first demonstration of the in vivo therapeutic efficacy of pharmacological STING activation against SADS-CoV. These findings demonstrate that the STING pathway serves as a critical regulator of host defense against SADS-CoV and suggest that STING-targeted intervention has therapeutic potential.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging zoonotic pathogen with significant implications for veterinary and public health; it has a high mortality rate in piglets and the potential for cross-species transmission. Currently, there are no approved vaccines or specific antiviral agents available for this pathogen. In this study, we demonstrated that the stimulator of interferon genes (STING) pathway serves as a critical mediator of host defense against SADS-CoV infection. STING activation inhibits viral replication by coordinating interferon responses and modulating NF-κB/IRF3 signaling, and its inhibition exacerbates infection. Importantly, pharmacological activation of the STING pathway using the agonist diABZI significantly inhibited viral replication in vivo in a STING-dependent manner, with contributions from both type I interferon-dependent and -independent antiviral mechanisms, highlighting its therapeutic potential. These results advance our understanding of antiviral defense strategies against SADS-CoV and identify STING pathway regulation as a viable therapeutic approach for this emerging pathogen.

Background: Getah virus (GETV) is a vector-borne virus that can proliferate in mosquitoes and be transmitted to host animals through bites. Clinical infection with the virus mainly causes diarrhea and reproductive disorders in pigs and fever, rash, and edema in horses. This caused huge losses to the pig industry and also affected the level of competition for horses. GETV has proliferated across over 20 regions within China, recently resulting in miscarriages and fatalities among the pig population, Guangdong province. Currently, there are no highly effective preventative or therapeutic strategies for diseases induced by GETV. Understanding the infection pathways, inclusive of GETV transmission vectors, is of paramount importance for the prevention and management of the disease. Results: To clarify the main transmission vectors and genotypes of GETV in this area, 3600 mosquitoes of different species were collected and GETV was detected using quantitative reverse transcription PCR. The minimum infection rate was 1.36 for Culex tritaeniorhynchus and 0.83 for Anopheles sinensis. The GETV GD2202 strain was successfully isolated from C. tritaeniorhynchus using mouse neuroblastoma (N2a) cells, and its complete genome was sequenced by PCR. This represents the first identification and isolation of GETV from mosquitoes in Guangdong Province of Southern China. Comparison with GenBank data showed 99.3% identity with the E2 gene of the GDFS2-2018 and GDFS9-2018 strains causing disease in local pig populations, and a nucleotide similarity of 99.8% with the E2 gene of the HNNY-1, HNPDS-1, and HNPDS-2 strains isolated from pigs in Henan, and the highest identity with the JL1708 strain isolated from mosquitoes in Jilin Province, with a nucleotide similarity in the E2 gene of 99.9%. Phylogenetic analysis showed that it had the closest genetic evolutionary relationship with the Culex-derived JL1708 strain and was on the same evolutionary branch as the pig-derived HNNY-1, HNPDS-1, and HNPDS-2 strains. Conclusion: A comprehensive investigation was undertaken to examine the prevalence of GETV infection among various mosquito species in Foshan, Guangdong Province, China. The findings indicated that C. tritaeniorhynchus acted as the principal vector for transmission, predominantly infected with GETV genotype III. This genotype was consistent with that identified in deceased pig populations and demonstrated significant homology. This study provides a robust scientific basis for understanding the propagation of GETV, thereby offering vital insights for the formulation of disease prevention and control strategies.

The Aedes albopictus C6/36 cell line is model system for studying mosquito-borne viruses. These cells exhibit high permissiveness to Japanese encephalitis virus (JEV), supporting robust viral replication and producing a significant viral load. Given C6/36 cells established susceptibility, this study employed 4D label-free quantitative proteomics to investigate the underlying mechanisms that enable C6/36 cells to be readily cultured and facilitate efficient JEV infection. C6/36 cells were infected with JEV at MOI of 0.5 and harvested at 0,6,12, and 24 hours post-infection (HPI) for 4D label-free quantitative proteomics analysis. Proteomic data analysis revealed that differentially expressed proteins were primarily involved in signal transduction mechanisms, innate immune responses, metabolism, and the synthesis, transport, and catabolism of secondary metabolites. JEV infection downregulated several proteins involved in DNA replication, including proliferating cell nuclear antigen (PCNA), DNA polymerase epsilon subunit 3 (POLε3), and DNA primase large subunit (PRI2), thereby inhibiting DNA replication signaling pathways and contributing to increased JEV replication. JEV infection also suppressed Toll-like receptors (TLRs), key components of the innate immune system. Specifically, TLR2, TLR4, TLR7, and TLR13 were significantly downregulated, thereby suppressing the innate immune response. Our results suggest that JEV infection impairs DNA replication and innate immune responses, particularly the inhibition of PRI2 and POLε3, leading to increased JEV replication and potentially a key mechanism of JEV pathogenesis. Elucidating the interactions between JEV and the host DNA replication and immune system will facilitate the development of more effective JEV prevention and treatment strategies.

Conventional influenza vaccine can prevent infection and reduce the risk of post-infection complications. However, they lack the capacity to effectively respond to influenza virus mutations. This results in the vaccine becoming ineffective due to a reduced antigenic match. It is necessary to develop a new strategy for vaccine that will provide broad cross-reactive protection. A DNA vaccine based on the hemagglutinin (HA) gene and conserved antigenic epitopes of both the HA, M2e and NA genes to provide protection against influenza B was developed. BALB/c mice were immunized with electroporation to evaluate both humoral immune responses and T cell responses. Protection against influenza B virus challenge was evaluated in DNA vaccinated mice, followed by analysis of lung tissue to assess changes in cytokine levels and virus load. Additionally, various assays with DNA were conducted to assess their cellular uptake by DCs and their potential for immune activation. Vaccine via electroporation demonstrated the ability to enhance both humoral and cellular immune responses and resulted in the shaping of the immune response to the vaccine in a Th1 direction. Animals inoculated with vaccines via electroporation were completely protected against both homologous and heterologous viruses, as evidenced by the reduction of lung viral loads and lung inflammation, induction of broadly cross-protective humoral immunity, and IL-2 CD4+ T-cell responses. The most significant finding was that the DNA vaccine provided complete protection for mice against two distinct lineages of the lethal influenza B virus. These findings suggested that DNA vaccine delivered using in vivo electroporation effectively elicits a protective immune response and provides additional cross-protection.

The recent global outbreaks of mpox highlight the urgent need for both fundamental research and antiviral development. However, studying monkeypox virus (MPXV), with its large and complex genome, remains challenging due to the requirement for high-containment facilities. Here, we describe a novel strategy for de novo assembly of MPXV clade IIb genomes in bacterial artificial chromosomes using transformation-associated recombination cloning. Leveraging CRISPR-Cas9 and Lambda Red recombination, we engineered replication-defective MPXV particles with dual deletions of OPG96 ( M2R ) and OPG158 ( A32.5L )—genes essential for virion assembly, that are capable of recapitulating key stages of the viral life cycle. Our work demonstrates the utility of replication-defective MPXV particles as a reliable platform for high-throughput antiviral discovery, offering significant advantages for both fundamental virology studies and therapeutic development against orthopoxviruses. ### Competing Interest Statement The authors have declared no competing interest.

Porcine idiopathic vesicular disease (PIVD), one of several clinically indistinguishable vesicular diseases of pigs, is caused by the emerging pathogen Senecavirus A (SVA). Despite the widespread prevalence of porcine SVA infection, no effective commercial vaccines for PIVD prevention and control are available, due to high costs associated with vaccine testing in pigs, considerable SVA diversity, and SVA rapid evolution. In this study, SVA CH/JL/2022 (OP562896), a novel mutant SVA strain derived from an isolate obtained from a pig farm in Jilin Province, China, was inactivated then combined with four adjuvants, MONTANIDETM GEL02 PR (GEL 02), MONTANIDETM ISA 201 VG (ISA 201), MONTANIDETM IMG 1313 VG N (IMS1313), or Rehydragel LV (LV). The resulting inactivated SVA CH/JL/2022 vaccines were assessed for efficacy in mice and found to induce robust in vivo lymphocyte proliferation responses and strong IgG1, IgG2a, and neutralizing antibody responses with IgG2a/IgG1 ratios of <1. Furthermore, all vaccinated groups exhibited significantly higher levels of serum cytokines IL-2, IL-4, IL-6, and IFN as compared to unvaccinated mice. These results indicate that all vaccines elicited both Th1 and Th2 responses, with Th2 responses predominating. Moreover, vaccinated mice exhibited enhanced resistance to SVA infection, as evidenced by reduced viral RNA levels and SVA infection-induced histopathological changes. Collectively, our results demonstrate that the SVA-GEL vaccine induced more robust immunological responses in mice than did the other three vaccines, thus highlighting the potential of SVA-GEL to serve an effective tool for preventing and controlling SVA infection.

Porcine teschovirus (PTV) can cause reproductive dysfunction and respiratory diseases, with high mortality rates for sick pigs. Among Picornaviridae family virus-encoded proteins, the VP1 structural protein is critical for viral immune evasion. However, whether PTV VP1 inhibits the type I interferon (IFN) response remains unknown. Here, it shows that the PTV VP1 protein significantly hinders the activation of NF-κB, impairing Sendai virus-induced expression of beta IFN (IFN-β). Further studies revealed that VP1 targets and interacts with the MDA5 factor in the RIG-I like receptors pathway. More importantly, the VP1 protein interacts with the caspase activation and recruitment domain and Hel domains of MDA5, blocking IFN-β expression. Our findings provide evidence about the VP1 protein of PTV hinders MDA5 activation and may represent a viral mechanism to escape the innate immune response.

PCV2 and PRRSV are involved in a variety of disease syndromes, collectively referred to as Porcine Circovirus Associated Disease (PCVAD) and Porcine Respiratory Disease Complex (PRDC). To explore the effects of other pathogens on porcine PCVAD and PRDC. This study firstly conducted an epidemiological investigation on PPV, confirmed the prevalence of PPV sub-types in China; We then determined that other pathogens were more common in POS-PCV2 (PCV2 positive other pathogen detection rate) than NEG-PCV2 (other pathogen detection rate PCV2 negative); however, in POS-PRRSV (PRRSV positive other pathogen detection rate), the detection rate was lower than that of NEG-PRRSV (other pathogens were negative for PRRSV). To study the effect of PPV2, PPV3, PPV4, PPV5, PPV6, PPV7, PRRSV, PCV2, PCV3, TTsuV1, and TTsuV2 co-infection on PCV2-associated disease (PCVAD) and porcine respiratory disease complex (PRDC), we used a logit-link and a generalized linear model (GLM) of the binomial error distribution and compute the predictions. PCV3 (P < 0.001***), PRRSV (P < 0.001***), and TTsuV2 (P < 0.001***) were predicted to be the most significant factors associated with PCV2 in PCVAD and PRDC, PCV2 (P < 0.001***), PCV3 (P < 0.001***) and TTsuV2 (P = 0.003**) were the most significant factors associated with PRRSV in PCVAD and PRDC. These findings determine that PPV2, PPV3, PPV4, PPV5, PPV6, PPV7, PRRSV, PCV2, PCV3, TTsuV1, and TTsuV2 viruses are statistically associated as co-infectors frequency with PCV2 in PRDC and PRRSV in PCVAD diseased pigs through logit-link and a generalized linear model (GLM) of the binomial error distribution.