Development of Porcine Monoclonal Antibodies with In Vitro Neutralizing Activity against Classical Swine Fever Virus from C-Strain E2-Specific Single B Cells.

Neutralizing antibodies (nAbs) can be used before or after infection to prevent or treat viral diseases. However, there are few efficacious nAbs against classical swine fever virus (CSFV) that have been produced, especially the porcine-originated nAbs. In this study, we generated three porcine monoclonal antibodies (mAbs) with in vitro neutralizing activity against CSFV, aiming to facilitate the development of passive antibody vaccines or antiviral drugs against CSFV that offer the advantages of stability and low immunogenicity. Pigs were immunized with the C-strain E2 (CE2) subunit vaccine, KNB-E2. At 42 days post vaccination (DPV), CE2-specific single B cells were isolated via fluorescent-activated cell sorting (FACS) baited by Alexa Fluor™ 647-labeled CE2 (positive), goat anti-porcine IgG (H + L)-FITC antibody (positive), PE mouse anti-pig CD3ε (negative) and PE mouse anti-pig CD8a (negative). The full coding region of IgG heavy (H) chains and light (L) chains was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Overall, we obtained 3 IgG H chains, 9 kappa L chains and 36 lambda L chains, which include three paired chains (two H + κ and one H + λ). CE2-specific mAbs were successfully expressed in 293T cells with the three paired chains. The mAbs exhibit potent neutralizing activity against CSFVs. They can protect ST cells from infections in vitro with potent IC50 values from 14.43 µg/mL to 25.98 µg/mL for the CSFV C-strain, and 27.66 µg/mL to 42.61 µg/mL for the CSFV Alfort strain. This study is the first report to describe the amplification of whole-porcine IgG genes from single B cells of KNB-E2-vaccinated pig. The method is versatile, sensitive, and reliable. The generated natural porcine nAbs can be used to develop long-acting and low-immunogenicity passive antibody vaccine or anti-CSFV agents for CSF control and prevention.

Assessment of regional vulnerability to Africa swine fever in China during 2018/8-2019/7 based on data envelopment analysis method.

In this study, we introduce a vulnerability index to measure the regional ASF epidemic and present the ASF severity ratings of the 31 provinces of mainland China. The index is defined based on the data from the investigation, national statistical yearbook and reports. The data to be used include pig breeding, financial resources, human resources, epidemic information of ASF and price fluctuation from the 31 provinces. Then, we use the data envelopment analysis (DEA) method to define the vulnerability index, the relative severity value for each region, which quantitatively reflects the damage degree caused by the epidemic of ASF. The method allows us to provide a systematic classification for the regional vulnerability level of ASF in China. Using this index, we find that the vulnerability of the whole country is at a high level, and there is no regional aggregation phenomenon. The vulnerability level of the 31 provinces is quite different and the provinces with high vulnerability level are dispersive geographically. For the five major prevention and control zones for ASF in China, the northern region has the highest vulnerability level, while the eastern zoon level is the lowest.

Rapid visual CRISPR assay: a naked-eye colorimetric detection method for nucleic acids based on CRISPR/Cas12a and convolutional neural network (preprint)

Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with CRISPR/Cas12a based visual colorimetric assays. Among them, 9 ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for naked-eye read-out of assay results (no transducing component required for visualization). Subsequently, we developed a convolutional neural network algorithm standardize and to automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube towards an instrument-less colorimetric POCT format that requires only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the single-copy detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this novel RAVI-CRISPR system for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen-side testing.

Integrating digital and field surveillance to complement efforts to manage epidemic diseases of livestock: African swine fever as a case study (preprint)

The SARS-CoV-2 pandemic has unveiled the importance of stakeholders and ordinary citizens in managing infectious disease emergencies. Efficient management of infectious diseases requires a top-down approach which must be complemented with a bottom-up response to be effective. Here we investigate a novel approach to surveillance for transboundary animal diseases using African Swine fever as a model. We were able to collect data at a population level on information-seeking behavior and at a local level through a targeted questionnaire-based survey to relevant stakeholders such as farmers and veterinary authorities. Our study shows how information-seeking behavior and resulting public attention during an epidemic, can be addressed through novel data streams from digital platforms such as Wikipedia. We also bring evidence on how field surveys aimed at local workers (e.g. farmers) and public authorities remain a crucial tool to assess more in-depth preparedness and awareness among front-line actors. We conclude that they should be used in combination to maximize the outcome of surveillance and prevention activities for selected transboundary animal diseases.

Impacts of Coronavirus on Farm and Pet Animals (preprint)

Coronaviruses are positive sense RNA virus belonging to the Coronaviridae family, which are further subdivided into four genera: Alpha, Beta, Gamma, and Delta Coronaviruses. Infectious bronchitis virus and SARS-CoV belong to Beta Coronaviridae family. Infectious bronchitis virus causes respiratory and nephritic signs that includes tracheal rales, urate crystals, lethargy and nasal discharge. In livestock and pets, the Coronavirus infection causes mostly gastrointestinal lesions, which may be prevented through vaccination and biosecurity. Recent infections of SARS-CoV-2 (also known as COVID-19) on farm and pet animals were summarized in this study. Besides, zoo animals were reported with infections in some countries/regions. Although the damage of COVID-19 has not been reported as serious as highly pathogenic avian influenza (HPAI) and African Swine Fever (ASF) on farm animals so far, the transmission mechanism of COVID-19 among group animals/farms and its long-term impacts are still not clear. The impact of Coronavirus on animals and potential prevention strategies, such as vaccine development and farm biosecurity measures, were discussed. Prior to the development of the effective vaccine, the biosecurity measures (e.g., conventional disinfection strategies and innovated technologies) may play roles in preventing potential spread of diseases/viruses.