Porcine deltacoronavirus nonstructural protein 2 inhibits type I and III IFN production by targeting STING for degradation.

Porcine deltacoronavirus (PDCoV) is an enteropathogenic coronavirus that has been reported to use various strategies to counter the host antiviral innate immune response. The cGAS-STING signalling pathway plays an important role in antiviral innate immunity. However, it remains unclear whether PDCoV achieves immune evasion by regulating the cGAS-STING pathway. Here, we demonstrated that the nonstructural protein 2 (nsp2) encoded by PDCoV inhibits cGAS-STING-mediated type I and III interferon (IFN) responses via the regulation of porcine STING (pSTING) stability. Mechanistically, ectopically expressed PDCoV nsp2 was found to interact with the N-terminal region of pSTING. Consequently, pSTING was degraded through K48-linked ubiquitination and the proteasomal pathway, leading to the disruption of cGAS-STING signalling. Furthermore, K150 and K236 of pSTING were identified as crucial residues for nsp2-mediated ubiquitination and degradation. In summary, our findings provide a basis for elucidating the immune evasion mechanism of PDCoV and will contribute to the development of targets for anti-coronavirus drugs.

An Efficient and Rapid Assay for Detecting Neutralizing Antibodies Against Serotype 4 Fowl Adenovirus.

Currently, the outbreak of serotype 4 fowl adenovirus (FAdV-4) has spread worldwide and caused tremendous economic loss to the poultry industry. Although inactivated vaccines have been licensed against FAdV-4 in China, a rapid and efficient serological method for measuring the titer of neutralizing antibodies (NAbs) specific for FAdV-4 post-infection or vaccination is rarely reported. Classical virus neutralization test (VNT) is superior in sensitivity and specificity for detecting NAbs but is either time-consuming or laborious. In this study, a recombinant virus FA4-EGFP expressing EGFP-fiber-2 fusion protein, rather than wild type (WT) FAdV-4 was used to develop a novel VNT for detecting FAdV-4 NAbs. Specificity analysis showed that the approach only reacted with the sera against FAdV-4, not with the sera against other avian pathogens tested. The novel VNT was effective in the detection of NAbs against FAdV-4 in sera from both experimentally infected and clinically vaccinated chickens, and had good linear correlation with the classical VNT. Moreover, the novel VNT not only significantly simplifies the procedure for detection of NAbs, but also shortens the timeline to 24 h in comparison with the classical VNT with 3-4 d. All these data demonstrate that the FA4-EGFP based VNT developed here provides an efficient diagnostic method for monitoring the immunological state of the vaccination or diagnosing the clinical infection of FAdV-4 in a quick and funding-saving manner.

A Novel Recombinant FAdV-4 Virus with Fiber of FAdV-8b Provides Efficient Protection against Both FAdV-4 and FAdV-8b.

Since 2015, the outbreaks of hydropericardium-hepatitis syndrome (HHS) and inclusion body hepatitis (IBH) caused by the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) and serotype 8 fowl adenovirus (FAdV-8), respectively, have caused huge economic losses to the poultry industry. Although several vaccines have been developed to control HHS or IBH, a recombinant genetic engineering vaccine against both FAdV-4 and FAdV-8 has not been reported. In this study, recombinant FAdV-4 expressing the fiber of FAdV-8b, designated as FA4-F8b, expressing fiber of FAdV-8b was generated by the CRISPR-Cas9 and homologous recombinant techniques. Infection studies in vitro and in vivo revealed that the FA4-F8b replicated efficiently in LMH cells and was also highly pathogenic to 2-week-old SPF chickens. Moreover, the inoculation of inactivated the FA4-F8b in chickens could not only induce highly neutralizing antibodies, but also provide efficient protection against both FAdV-4 and FAdV-8b. All these demonstrate that the inactivated recombinant FA4-F8b generated here can act as a vaccine candidate to control HHS and IBH, and FAdV-4 can be an efficient vaccine vector to deliver foreign antigens.

An efficient peptide-based ELISA for differentiating fowl adenovirus 4-infected chickens from vaccinated chickens.

Fowl adenovirus serotype 4 (FAdV4), the causative agent of hepatitis-hydropericardium syndrome (HPS), has caused major economic losses to the poultry industry worldwide. Although inactivated vaccines have been deployed widely against FAdV4, a DIVA (differentiating infected from vaccinated animals) test specific for FAdV4 has not been available. We synthesized an immunogenic peptide, corresponding to regions 66-88 aa of the 22K nonstructural protein of FAdV4, and used the peptide as coating antigen to develop an indirect ELISA for a DIVA test specific to FAdV4. Specificity analysis showed that the ELISA only reacted with sera against FAdV4, and not with sera against other pathogens tested. Moreover, the ELISA could effectively differentiate FAdV4-infected chickens from vaccinated chickens. In a test of sera from experimentally infected chickens, the ELISA had 95% and 85% concordance with an indirect immunofluorescence assay (indirect IFA) and a commercial ELISA, respectively, and the concordance was 80.5% between the ELISA and the indirect IFA in detecting clinical infection samples. Our peptide-based ELISA provides an efficient DIVA test for FAdV4 in clinical samples.

Development of colloidal gold-based test strip for rapid detection of serotype 4 fowl adenovirus.

The outbreaks of hepatitis-hydropericardium syndrome (HPS) caused by serotype 4 fowl adenovirus (FAdV-4) have spread from broilers to laying hens, breeders, geese and Cherry Valley duck, resulting in high economic losses to the poultry industry globally. In this study, a rapid colloidal gold test strip for detection of FAdV-4 was developed by using two monoclonal antibodies (mAbs) against the Fiber-2 of FAdV-4. Specificity analysis revealed that the test strip only reacted with FAdV-4, but not with other pathogens including different serotypes of fowl adenovirus and other avian pathogens tested. The limit of the detection (LOD) of the strip could reach as low as 0.1 µg/0.1 mL of GST-Fiber-2 protein and 1 × 105 TCID50/0.1 mL of FAdV-4, respectively. Moreover, the test strip could be efficiently applied in detecting tissue samples from diseased chickens with HPS. Comparison analysis further revealed that the test strip showed good compatibility with PCR assay for detection of virus isolates and clinical samples. In conclusion, our test strip provides an efficient on-site diagnostic method in a quick and convenient manner for detection of FAdV-4, especially in resource-limited areas.

Long-term effect of soil and water conservation measures on runoff, sediment and their relationship in an orchard on sloping red soil of southern China.

The effect of soil and water conservation measures (SWCMs) is usually dependent on time. Thus the trend in reducing runoff and sediment over time is a very important theoretical problem for evaluating the effectiveness of SWCMs. Moreover, there is still a lack of comprehensive assessment of water erosion dynamics following implementing SWCMs despite their ecological significance. Therefore, the long-term impact of SWCMs on runoff and sediment and their relationships was assessed for an orchard on sloping red soil in southern China. Overland flow and erosion sediment were continuously observed for 15 years on citrus experimental plots under one of four treatments: grass strips, strip intercropping, level terrace and clean-tillage. By means of Mann-Kendall trend tests and double cumulative curves, the time series of runoff and sediment under the different treatments were analyzed. Furthermore, we linked the effect of soil conservation and the relationship between runoff and sediment variation to determine the mechanism of conservation measures on sediment reduction. The results showed that the first 4 years was the key period to prevent soil erosion for this orchard, and then the intensity of soil erosion decreased below 500 t·km-2·a-1. Considering economic costs and ecological effect, grass strips were the best protective measure for this test situation. The fitted curves of the effect of SWCMs on sediment reduction over time showed an 'L' form, but on runoff there was an approximately horizontal line. The SWCMs did not change the rainfall-runoff relationship, but did change the runoff-sediment erosion relationship. The erosion reduction mechanism of SWCMs in the early phase was a joint function of reducing runoff and changing the runoff-sediment relationship, and in the post-stable phase it worked mainly by reducing runoff. The results provide the basis for rational allocation of SWCMs considering location and time.