Abstract The whitefly Bemisia tabaci is an important vector of the devastating tomato yellow leaf curl virus (TYLCV). The widespread insecticide resistance issues, however, seriously challenge the current practices; novel vector management strategies targeting transmission pathways are urgently needed. In this study, we demonstrate that viral infection upregulates UDP‐Bta06664 , a UDP‐glycosyltransferase (UGT) gene in B. tabaci , resulting in elevated UGT activity. Silencing UDP‐Bta06664 via RNA interference (RNAi) significantly reduced TYLCV acquisition and inoculation efficiency while maintaining vector survival. The electrical penetration graph showed that UDP‐Bta06664 suppression disrupted key phloem‐feeding parameters, significantly delaying and shortening phloem salivation and sustained phloem ingestion. These behavioral alterations explain the observed reductions in virus uptake and delivery, establishing that UDP‐Bta06664 acts as a metabolic facilitator of virus transmission. This study highlights a metabolic–behavioral axis regulating plant virus transmission and identifies a potential molecular target for RNAi‐mediated disease control.

Adeno-associated virus (AAV) is a key vector in gene therapy, yet conventional AAV quality control methods are time-consuming and unsuitable for real-time monitoring, severely hindering process optimization. Here, we present an aldehyde-functionalized metasurface plasmon resonance (ALMetaSPR) biosensor for rapid, label-free, and high-throughput AAV quantification. Two complementary platforms were developed: a 96-well high-throughput system enabling analysis within 15 min (8-fold faster than ELISA); and a portable MetaSPR-based process analytical technology (PAT) device (MSPR-PAT) that integrates microfluidics and smartphone interfaces for real-time online monitoring of AAV production. Both platforms can accurately quantify AAV-2 and AAV-5 titers directly without sample labeling. The antibody-based capture system exhibited a broad dynamic range (6.25 × 1009-4.00 × 1011 GC/mL) with a limit of detection (LOD) of 1.24 × 1009 GC/mL, representing a 250-fold sensitivity improvement over current label-free techniques. The biosensor surface can be regenerated more than 42 cycles without performance loss, which significantly reduces the per-test costs. This innovation resolves a critical bottleneck in gene-therapy manufacturing by integrating high-throughput screening, real-time process analytics, and one-step operation.

Assessing intestinal barrier function in organ- and cell-on-a-chip models is crucial for studying intestinal diseases and advancing drug development. However, current technologies lack methods for continuous, non-destructive monitoring of intestinal barrier dynamics. In this study, we developed a real-time, label-free, high-throughput cell-on-a-chip system based on a metasurface plasmon resonance colorimetric sensor (MetaSPRCS) to evaluate intestinal barrier function. Integrating MetaSPRCS with transmission microscopy enabled real-time visualization of cell adhesion, proliferation, and intestinal barrier layer formation, disruption, and repair. The MetaSPRCS platform non-destructively detects subtle epithelial cell damage caused by low ethanol concentrations, with a two-fold higher sensitivity than that of CCK-8 assays. In an alcohol-induced intestinal barrier injury model, MetaSPRCS validated a reliable evaluation of Dihydroquercetin (DHQ), consistent with endpoint methods. Animal experiments confirmed its ability to predict in vitro-to-in vivo dose conversion of DHQ, offering promising guidance for the administration of novel cell-repairing food ingredients. This study provides a high-throughput, visual, non-destructive tool for assessing intestinal homeostasis, establishing a preliminary dose conversion relationship between in vitro evaluation experiments and in vivo animal administration.

Objective Alpha-fetoprotein (AFP) is a critical biomarker for diagnosing hepatocellular carcinoma (HCC) and other malignancies, as well as for prenatal screening. However, existing detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, and gold immunochromatographic assay suffer from limitations including low sensitivity, long detection time, complex operations, or inability to quantify. Traditional surface plasmon resonance technology, while powerful for biomolecular interaction analysis, is hindered by bulky equipment, high costs, and complicated data analysis. Thus, there is an urgent need for a high-performance detection platform that is sensitive, rapid, cost-effective, and suitable for high-throughput applications. This study aims to develop a novel metasurface plasmon resonance (MetaSPR) biosensor integrated with gold nanoparticles (AuNPs) labeling to enable both affinity evaluation of AFP antigen-antibody interactions and rapid quantitative detection of AFP in human serum, addressing the aforementioned limitations. Methods The MetaSPR biosensor is fabricated using advanced nanoimprint technology combined with laser interference lithography and ion etching, creating a 3D nanocup array structure on a polyethylene terephthalate (PET) substrate. The nanocup array is coated with a 15 nm titanium (Ti) layer, a 70 nm silver (Ag) layer, and a 20 nm gold (Au) layer via vacuum deposition to enhance plasmonic properties and stability. The sensor is integrated into a 96-well plate for high-throughput analysis. For AFP detection, the biosensor surface is functionalized by immobilizing AFP capture antibodies using 50 mmol/L CBS buffer (pH=9.6) and blocking with 1% casein to reduce non-specific binding. Affinity evaluation of AFP antigen-antibody interactions is performed using a direct capture method, monitoring binding and dissociation kinetics with a WeSPRTM 200 detector. Epitope identification of monoclonal antibodies (AFP mAb 1 and AFP mAb 2) is conducted via a tandem binding assay. For quantitative detection, AuNPs are conjugated with AFP detection antibodies under optimized conditions (4 & micro;g/mL antibody concentration) to amplify detection signals. A sandwich immunoassay is established using these functionalized sensors and AuNPs-labeled antibodies, a standard curve is constructed using AFP standards (0.5-32 ng/mL) with HBS-ET as the diluent, and its performance is validated through specificity tests (using prostate-specific antigen, PSA) and spiked recovery experiments in human serum. Results and Discussions The MetaSPR biosensor exhibits a highly uniform 3D nanocup array structure (Fig. 1), confirmed by scanning electron microscopy (SEM). Its plasmonic response is validated using CaCl2 solutions, showing a linear correlation between refractive index changes and optical signals (R-2=0.999), confirming high sensitivity to surface refractive index variations (Fig. 2). Affinity analysis reveals two dissociation constants (KD) for AFP antigen-antibody interactions: 6.81 & times;10(-10) mol/L (AFP mAb 1) and 2.25 & times;10(-10) mol/L (AFP mAb 2), indicating strong binding affinities. Epitope identification confirms that AFP mAb 1 and AFP mAb 2 recognize distinct epitopes, enabling their use in a sandwich assay. The optimal antibody pairing (AFP mAb 1 as capture antibody and AFP mAb 2 as detection antibody) is validated via absorbance spectral shifts (Fig. 3). For quantitative detection, the AuNPs-amplified sandwich assay achieves a linear standard curve in the range of 0.5-32 ng/mL (R-2=0.997) with a limit of detection (LOD) of 0.2 ng/mL (Fig. 6), which is 10-fold more sensitive than traditional ELISA. The total detection time is reduced to 15 min, 16 times faster than ELISA. Spiked recovery experiments in human serum (1: 20 and 1: 40 dilutions) yield recoveries of 82% -120% (Table 1), confirming accuracy. Specificity tests show no cross-reactivity with PSA, demonstrating high selectivity for AFP (Fig. 5). Conclusions This study successfully develops a MetaSPR biosensor for affinity evaluation and quantitative detection of AFP. This platform can rapidly and accurately achieve affinity evaluation (with KD values of 6.81 & times;10(-10)mol/L and 2.25 & times;10(-10) mol/L, respectively) and can perform rapid quantitative analysis of AFP in human serum. Utilizing AuNPs labeling technology, it has an LOD of 0.2 ng/mL, a detection time of 15 min, and high-throughput capability (96 samples detectable per run). Its superior performance- including enhanced sensitivity, speed, and cost-effectiveness-overcomes the limitations of conventional methods. The biosensor's specificity, accuracy, and ease of use make it a promising tool for early diagnosis and monitoring of HCC and other AFP-related diseases, with potential for broader application in clinical and research settings. Future work will focus on expanding its utility to other biomarkers and advancing clinical translation.

BACKGROUND:Severe fever with thrombocytopenia syndrome (SFTS), caused by the severe fever with thrombocytopenia syndrome virus (SFTSV), poses a serious public health threat due to its high fatality rate, and delayed diagnosis is strongly associated with disease progression and increased mortality. Conventional laboratory diagnosis still relies largely on quantitative real-time PCR (qPCR), which requires specialized instruments, trained personnel, and centralized testing facilities. These challenges underscore the urgent need for rapid, sensitive, and portable diagnostic methods for SFTSV detection in clinical and on-site settings. RESULTS:Here, we developed an integrated isothermal amplification-based point-of-care testing (IAPOCT) platform that incorporates a recombinase polymerase amplification (RPA)-based sensing and interpretation strategy with a smartphone-interfaced portable device. This platform demonstrated low-copy analytical detection capability, achieving a limit of detection (LOD) as low as 10 copies/μL. The core amplification and fluorescence readout steps can be completed within 20 min, while the complete serum testing workflow from thermal lysis to result interpretation requires approximately 25 min. Preliminary evaluation in a cohort of 24 clinical serum specimens showed complete qualitative agreement with qPCR, correctly identifying 20 positive and 4 negative samples. Endpoint fluorescence signals were analyzed through the smartphone-based interface, enabling portable and objective qualitative interpretation of clinical samples. The lyophilized reagent format was incorporated to facilitate handling and integration with the portable testing workflow for routine on-site molecular testing in resource-limited settings. SIGNIFICANCE:This work provides a practical platform for rapid SFTSV detection with portable fluorescence readout, with potential applications in clinical screening and on-site epidemiological surveillance. By integrating RPA-based amplification, lyophilized reagents, smartphone-assisted interpretation, and a portable device, the platform offers a compact analytical strategy for point-of-care molecular testing of infectious diseases.

Bemisia tabaci is a globally important crop pest and virus vector with widespread resistance to conventional insecticides. We evaluated the insecticidal activity of a soybean-derived lecithin preparation and associated physiological responses. Leaf-surface, dried-residue contact, artificial-diet, and egg bioassays were combined with RNA sequencing, untargeted metabolomics, RT-qPCR, histology, and transmission electron microscopy. Dried-residue exposure yielded a 4 h LC50 of 1.16 mg mL−1 (95% confidence interval, 0.948–1.403 mg mL−1), whereas 10 mg mL−1 lecithin caused 100% adult mortality within 4 h and reduced egg hatchability from 99.44% to 37.78%. No mortality was detected after adults were provided with a sucrose diet containing 10 mg mL−1 lecithin for 24 h. Exposure at the 4 h LC50 was associated with differential expression of 135 genes and changes in 599 metabolic features, including responses related to cuticle organization, lysosomal and autophagy pathways, and amino acid metabolism. Histology and transmission electron microscopy revealed altered abdominal tissue organization and cellular ultrastructure. These findings establish contact-associated and egg-stage activity of lecithin against B. tabaci under laboratory conditions and identify accompanying structural, transcriptional, and metabolic responses. The molecular events responsible for mortality and the contribution of ingestion-mediated exposure remain unresolved.

Tumor cell surface sialoglycans interact with Siglec receptors to mediate immunosuppression, representing a promising therapeutic target. Here, we constructed an engineered bacterium (eSLC-Sia) that inducibly expresses sialidase and triggers drug release via quorum-mediated lysis. By modifying its surface with doxorubicin (DOX), we constructed a novel tumor-targeted bacterial-drug hybrid (eSLC-Sia@Dox). The system was demonstrated to rapidly form a protein corona from serum proteins, enabling evasion of immune recognition and facilitating controlled drug release within the acidic tumor microenvironment (pH 6.5), efficiently promoting the intratumoral penetration and distribution of DOX and sialidase, further inducing immunogenic cell death (ICD) effects. In 4 T1 tumor-bearing mice, eSLC-Sia@Dox achieved potent tumor suppression with minimal toxicity, increased tumor apoptosis (Bax/Bcl-2 ratio and cleaved caspase-3), promoted HMGB1 release indicative of immunogenic cell death, and enhanced immune infiltration (CD45+ and CD8+ cells). These findings underscore the therapeutic potential of biohybrids in enhancing the synergistic effects of sialidase-mediated immunotherapy and DOX-triggered immunogenic chemotherapy, offering novel delivery strategies to overcome the immunosuppressive barriers of solid tumors.

Sepsis, a life-threatening organ dysfunction caused by infection, remains a major clinical challenge due to its high mortality and the urgent need for rapid, accurate diagnosis. Herein, we report a real-time metasurface plasmon resonance (MetaSPR) biosensor designed for sepsis diagnosis, overcoming the critical limitation of single-time point detection in conventional methods. Unlike ELISA or LAT, which only provide static detection snapshots, our real-time detection significantly reduced false positives, especially for low-concentration targets and in low signal-to-noise ratio situations. The real-time measurements combined with curve fitting can clearly distinguish the concentration of the detection point, improving the reproducibility, consistency, accuracy and resistance to interference of the results. This innovative platform integrates real-time detection with a rapid, highly sensitive, and high-throughput detection system. By optimizing nano-imprint parameters to control nanocup dimensions, we improved the sensor's sensitivity. The integration of AuNPs-labeled antibodies enables high-throughput, realtime quantitative detection of sepsis biomarkers on the chip surface, achieving a remarkable LOD of 39 pg/mL. The quantitative analysis of 80 clinical plasma samples shows excellent correlation (R2 = 0.899) with the latex turbidimetric method, highlighting the practical applicability of our high-throughput detection platform. This high-throughput, real-time biosensing system represents a significant advancement in clinical diagnostics with important scientific and clinical implications.

Tomato chlorosis virus (ToCV), transmitted by whiteflies (Bemisia tabaci), threatens global tomato production. However, the molecular mechanisms underlying the ToCV transmission by whiteflies remain largely unknown. Previous studies have shown that several key regulators of glycometabolism, downstream of the phosphoinositide 3-kinase (PI3K) pathway, are significantly increased in B. tabaci MED infected with ToCV. We demonstrate that ToCV activates PI3K in whiteflies, as evidenced by upregulated PI3K gene expression and increased PI3K enzyme activity. RNA interference-mediated knockdown of PI3K significantly reduced ToCV acquisition, retention, and transmission. Similarly, feeding whiteflies with the PI3K inhibitor PI3K-IN-1 decreased ToCV transmission, while the PI3K activator Recilisib enhanced it. Further analysis showed that the activation of PI3K enhances glycogen synthesis by upregulating genes related to glycogen synthesis, including serine/threonine kinase (AKT), glycogen synthase (GS), and glycogenin (Gly), and thus leads to an increase in glycogen content while reducing glucose levels. Conversely, the inhibition of PI3K disrupts glycogen metabolism, consequently impairing viral transmission. These findings highlight that PI3K is crucial for facilitating ToCV transmission by whiteflies and offer a potential target for controlling vector-borne plant viruses.

Surface plasmon resonance (SPR) technology has become a powerful tool for studying molecular interactions due to its label-free, real-time detection capabilities. However, the sensitivity limitations of conventional SPR have restricted its broader application. In response to this challenge, our study introduces an optical optimization, the photoluminescent-boosted Metasurface plasmon resonance (PLMLMSPR), which integrates photoluminescent molecule materials with a multilayer metal-enhanced metasurface plasmon resonance (MLM-MetaSPR) chip. Building upon the optimization of the nanocup array structure and multilayer metal composition, the metasurface chip is modified with a three-dimensional scaffold of carboxymethyl chitosan via carbene photopolymerization, further enhancing both protein immobilization and sensor sensitivity. Leveraging the metasurface chip, the photoluminescent molecule is labeled onto the antibody for signal amplification through a sandwich immunoassay system, achieving the ultra-sensitive PLMLMSPR platform. The incorporation of photoluminescent materials successfully harnessed nonradiative energy transfer to augment the local free electric field through enhanced transmission by absorbance of a photon, thereby significantly enhancing the plasmonic resonance effect in MetaSPR technology. This approach has doubled the sensor's response signal and improved the detection limit fourfold while maintaining protein-ligand affinity, as demonstrated by the effective detection of both SARS-CoV-2 wild-type and emerging variants, highlighting its remarkable practicality in diagnostic applications.

Two-dimensional MXene materials have gained attention in optics due to their excellent conductivity and light absorption, showing great potential in applications such as photodetectors, photothermal therapy, and laser protection. However, the application of MXene in enhancing plasmonic biosensing on metasurfaces has remained largely unexplored. The MXene functional metasurface plasmonic sensor (MetaSPR), integrated with the advanced microfluidic technology (3MSPR device), enables real-time monitoring of antibodies in three critical fields: precision diagnostics, drug development, and expression process monitoring. The MXene-MetaSPR, retaining the layered structure and high conductivity of MXene, exhibits enhanced sensitivity through its synergistic interaction with the electromagnetic fields within nanoarrays. The sensitivity of label-free IgG detection is increased by more than 250-fold, with a detection limit of 2.56 ng/mL. Experimental results demonstrate that this system is capable of detecting high-affinity (pM) binding between therapeutic antibodies and their targets. Additionally, it enables continuous monitoring during the nanobody expression process in engineered bacteria, with a single antibody concentration analysis completed within a time scale of minutes. The 3MSPR device rapidly and efficiently captures the initial phase of kinetic curves, facilitating precise affinity analysis and optimization of expression efficiency.

Tomato yellow leaf curl virus (TYLCV) poses a significant threat to tomato production, leading to severe yield losses. The current control strategies primarily rely on the use of pesticides, which are often nonselective and costly. Therefore, there is an urgent need to identify more environmentally friendly alternatives. Dufulin, a novel compound that has been effective in controlling viral diseases in tobacco and rice, has not yet been tested against TYLCV. This study assessed the efficacy of dufulin in controlling TYLCV over a three-year span from 2021 to 2023 through field trials, by monitoring disease symptoms and viral titers. Additionally, this study assessed the expression levels of genes associated with systemic acquired resistance (SAR), specifically proteinase inhibitor II (PI II) and non-expressor of pathogenesis-related genes 1 (NPR1), using real-time qRT-PCR. The chlorophyll and nitrogen content in the leaves were also measured. Plants treated with dufulin showed reduced symptomatology and lower viral titers compared to the controls. Analysis of gene expression revealed that NPR1 was upregulated in the dufulin-treated plants, whereas PI II expression was consistently downregulated in the TYLCV-infected plants. Interestingly, PI II expression increased in the healthy plants following a seven-day post-treatment with dufulin. Moreover, the treated plants exhibited a higher chlorophyll content than the controls, though no significant differences in the nitrogen levels were observed between the dufulin-treated and water-treated plants. Overall, the application of dufulin significantly bolstered the plant’s defense response, effectively reducing TYLCV symptoms and enhancing resistance.

The pursuit of biopharmaceutical in vitro efficacy evaluation has been significantly hindered by the lack of sensitive detection technologies, particularly in the field of high-affinity drug discovery and development. In this study, we developed a novel biosensor—zero-loss semiconductor materials-enhanced ultrasensitive metasurface plasmon resonance (ZLSMSPR). A metasurface was constructed based on the nanocup array structure of the ZLSMSPR chip, and light loss was minimized due to the zero-extinction coefficient property of Nb2O5, thereby amplifying the local SPR effect of the chip and enhancing refractive index sensitivity by 30 times. Additionally, we successfully optimized the grafting density of carboxylated dextran on the ZLSMSPR chip. This enhancement improved the immobilized ligand loading capacity, resulting in an impressive limit of detection (LOD) of 0.018 μg/mL. Furthermore, the ZLSMSPR platform was effectively applied to accurately evaluate the affinity of antibody-drug conjugates (ADCs) to their tumor targets and to closely monitor changes in affinity changes before and after the conjugation of cytotoxic drugs to the antibody. Notably, the developed ZLSMSPR biosensor demonstrated the capability to evaluate high-affinity interactions, with a sensitivity as low as 44.5 pM, underscoring the platform's potential for efficient, label-free, and highly sensitive in vitro evaluations.

The pursuit of cutting-edge diagnostic systems capable of detecting biomarkers with exceptional sensitivity and precision is crucial for the timely and accurate monitoring of inflammatory responses. In this study, we introduce a dual gold nanoparticle-enhanced metasurface plasmon resonance (Bi-MSPR) biosensor for the ultrasensitive detection of C-reactive protein (CRP). The Bi-MSPR sensor is constructed upon a nanocup array chip with gradient-free electron density, where an innovative metasurface structure is built using a PEI-immobilized dual-gold nanoparticle amplification system. This strategy greatly amplifies the local free electric field, resulting in a signal enhancement exceeding 10-fold, with a detection limit of 0.04 ng/mL for CRP in buffer solutions and 0.017 μg/mL for serum samples. Clinical validation demonstrates a 95.83% detection rate with no cross-reactivity, highlighting the sensor's remarkable specificity and sensitivity for CRP detection. The Bi-MSPR biosensor thus represents a rapid, sensitive, and specific diagnostic tool for CRP detection.

Investigating molecular interactions is crucial for advancing biological research and therapeutic discovery. Traditional analytical techniques often face limitations in sensitivity, quantification accuracy, and simplicity. Metasurfaces support resonances that are widely explored both for far-field wavefront shaping and for near-field sensing. Here, we introduce an innovative inverse-designed multilayer metasurface plasmon resonance (IDMM-SPR) sensor that overcomes these challenges. Using a double-objective optimization method that combines numerical simulations and machine learning, we developed an IDMM-SPR sensor featuring an optimized periodic nanocup array. This design yields unparalleled sensitivity and stability by harnessing collective resonances—including localized SPR, Wood’s anomalies, and the Bloch wave SPR—which collectively enhance the sensing performance to enable the analysis of ultra-high affinity and low molecular weight interactions. The sensor achieves a figure of merit (FoM) of 26.3 and a detection limit (LOD) for C-reactive protein (CRP) as low as 0.84 ng/mL. Its compatibility with microplate absorbance readers and imaging detection makes it highly practical. The IDMM-SPR sensor shows exceptional promise for high-throughput direct molecular fishing, drug development, and disease diagnosis, offering a powerful tool for real-time, label-free affinity detection and quantitative analysis of biomolecular interactions.

Pseudorabies (PR), caused by the PR virus (PRV), is an acute infectious disease in livestock and various wild animals. PRV has developed several immune evasion mechanisms to antagonize the host's antiviral immune response. However, the precise role of PRV-encoded proteins in regulating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon (IFN) gene (STING) signal for immune evasion remains unclear. In this study, we demonstrate that the PRV UL42 protein (pUL42) inhibited cGAS-mediated antiviral signaling by modulating cGAS recognition of dsDNA. Mechanistically, pUL42 interacts with the DNA-binding domain of cGAS, thereby inhibiting its recognition of double-stranded DNA, leading to the inhibition of its dimerization and oligomerization activation. Furthermore, knocking down the expression of the Ul42 gene in the PRV genome diminishes the antagonistic effect on type I IFN production and inhibits PRV replication. Ultimately, we have established that pUL42 targets cGAS-mediated signaling, thereby inhibiting the production of type I IFN and facilitating viral replication. Overall, our findings suggest that PRV pUL42 functions as an antagonist to evade the host's antiviral response by targeting the cGAS-STING axis. IMPORTANCE:Cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) axis is essential for host resistance to DNA virus infections by regulating type I interferon production. However, whether pseudorabies virus (PRV) antagonizes the cGAS-STING signaling pathway to immune evasion is not fully investigated. In this study, we clearly demonstrated that the PRV pUL42 protein inhibits the recognition of double-stranded DNA of cGAS, leading to inhibiting the oligomerization and activation of cGAS, thereby suppressing the cGAS-mediated host antiviral immune responses. Taken together, our results reveal a novel strategy employed by PRV to evade host defenses, which will provide theoretical support for the development of anti-PRV drugs for the prevention and control of PRV.

The accumulation of trace amounts of certain small molecules in food poses considerable human health challenges, including the potential for carcinogenesis and mutagenesis. Here, an ultrasensitive gold-platinum nanoflower-coupled metasurface plasmon resonance (MetaSPR) (APNMSPR) biosensor, based on a competitive immunoassay, was developed for the multiplexed and rapid quantitative analysis of trace small molecules in eggs, offering timely monitoring of food safety. This one -step biosensor can be integrated into either a newly designed detachable high-throughput MetaSPR chip-strip plate device or a standard 96-well plate for multiplexed small-molecule detection within a single egg. The limits of detection were 0.81, 1.12, and 1.74 ppt for florfenicol, fipronil, and enrofloxacin, respectively, demonstrating up to 1000-fold increased sensitivity and a 15-fold reduction in analysis time compared with those of traditional methods. The results obtained using the APNMSPR biosensor showed a strong correlation with those obtained using liquid chromatography-tandem mass spectrometry. The APNMSPR biosensor holds immense promise for the multiplexed, highly sensitive, and rapid quantitative analysis of small molecules for applications in food safety control, early diagnosis, and environmental monitoring.

IntroductionPorcine circovirus type 2 (PCV2) is the pathogen of Porcine Circovirus Associated Diseases. Porcine circovirus type 3 (PCV3) is a novel porcine circovirus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2 is clearly pathogenic, while the pathogenicity of PCV3 remains controversial, so it is crucial to monitor the prevalence of PCV2 and PCV3 in healthy and diseased pigs to investigate the effects of PCV3 and PCV2 on the health status of pigs.MethodsHere, we developed a PCV2 and PCV3 dual TaqMan quantitative PCR (qPCR) method to test samples from healthy and diseased pigs, to clarify the differences in the positive rates and viral copy numbers of PCV2 and PCV3, and to analyze the genetic evolution and molecular characterization of the viral genomes obtained with sequence alignment and phylogenetic analysis, homology and structural analysis of Cap proteins, and selection pressure analysis.ResultsWe successfully established a dual TaqMan qPCR method for PCV2 and PCV3 with good repeatability, specificity and sensitivity. In total, 1,385 samples from 15 Chinese provinces were tested with the established qPCR. The total positive rates were 37.47% for PCV3 and 57.95% for PCV2, and the coinfection rate for was 25.49%. The positive rates of PCV3 and PCV2 in 372 healthy pigs were 15.05 and 69.89%, respectively, and the coinfection rate was 12.90%. The positive rates of PCV3 and PCV2 in 246 diseased pigs were 55.69 and 83.33%, respectively, and the coinfection rate was 47.97%. Eighteen PCV3 genomes and 64 PCV2 genomes were identified, including nine each of the PCV3a-1 and PCV3b genotypes, eight of PCV2a, 16 of PCV2b, and 40 of PCV2d. The amino acid identity within the PCV3 Cap proteins was 94.00–100.0%, whereas the PCV2 Cap proteins showed an identity of 81.30–100.0%. PCV3 Cap was most variable at amino acid sites 24, 27, 77, 104 and 150, whereas PCV2 Cap had 10–13 unique sites of variation between genotypes.DiscussionThese results clarify the prevalence and variations of PCV2 and PCV3 in healthy and diseased pigs, which will provide a basis for the prevention and control of the two viral infections.

Porcine circovirus type 2 (PCV2) causes postweaning multisystemic wasting syndrome in piglets. Differences in the infectivity and horizontal transmissibility of different isolates of PCV2a, PCV2b, and PCV2d in pigs were evaluated by HE and IHC staining, PCR, virus titration, and IPMA to determine their clinical symptoms, pathological changes, levels of virus and antibody, and cohabitation infectivity. In the cohabitation infection experiment, weak viremia and low levels of antibodies were detected in the pigs challenged with PCV2a-CL, whereas no viremia or antibodies were detected in the corresponding cohabiting pigs. Furthermore, no PCV2 was isolated from any organ of pigs that were challenged with PCV2a-CL, as well as from those of their cohabiting pigs. In contrast, persistent viremia and pathological changes, including swollen inguinal lymph nodes, were detected in both the challenged and cohabiting pigs after PCV2b-BY or PCV2d-LNHC infection. Alive PCV2 was detected in the tonsils, inguinal lymph nodes, spleen, and kidneys of the experimental pigs by virus titration, and the highest viral titer was detected in the tonsils, followed by the inguinal lymph nodes. In a comparative analysis of the challenged and cohabiting pigs, a 1-week delay in viremia and specific antibodies was observed in the cohabiting pigs. Moreover, the number of viruses isolated from the tonsils and inguinal lymph nodes of the pigs cohabiting with PCV2d-LNHC-challenged pigs was significantly greater than those in the pigs that were directly challenged with PCV2d-LNHC in cohabitation infection experiment (P<0.05). Together, these results indicated that the infectivity and horizontal transmissibility of the strains PCV2b-BY and PCV2d-LNHC were much greater than those of the strain PCV2a-CL and provided some insights into PCV2 pathogenicity.