scholarly journals Development of a reverse transcription recombinase polymerase amplification assay with lateral flow dipstick for rapid detection of classical swine fever virus

2020 ◽  
Author(s):  
Jindai Fan ◽  
Yuanyuan Zhang ◽  
Wenxian Chen ◽  
Jingyuan Zhang ◽  
Chenchen Liu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Professional Staff ◽  
Lateral Flow ◽  
Fever Virus ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Simple Method ◽  
Lateral Flow Dipstick

Abstract Background: Classical swine fever (CSF), caused by the infection of Classical swine fever virus (CSFV), is a highly contagious disease of pigs and has caused significant economic losses in the pig industry. The rapid and effective detection of CSFV would contribute to the eradication program against CSF. Thus, this study aimed to develop a rapid and simple method for CSFV detection.Results: Here, a new method based on reverse transcription recombinase polymerase amplification (RT-RPA) coupled with lateral flow dipstick (LFD) was established for detecting CSFV. The RPA assay could be completed within 20 min at 37oC and the results of the RPA assay could be visualized by LFD assay with the naked-eye inspection. This RT-RPA-LFD assay could be used to detect CSFV specifically, with no cross-reaction with other pathogens. Its detection limit was 10 pg of CSFV cDNA. Importantly, the RT-RPA-LFD assay has a good performance on the CSFV detection for clinical samples.Conclusions: The established RT-RPA-LFD assay greatly reduced the need for professional staff and sophisticated instruments and made disease detection convenient and feasible. This method would be useful for the prevention and control of CSF, especially in resource-limited settings.

scholarly journals Identification of E2 with improved secretion and immunogenicity against CSFV in piglets

2020 ◽  
Author(s):  
Huiling Xu ◽  
Yanli Wang ◽  
Guangwei Han ◽  
Weihuan Fang ◽  
fang he
Keyword(s):  
Single Dose ◽  
Signal Peptide ◽  
Classical Swine Fever Virus ◽  
Subunit Vaccine ◽  
Classical Swine Fever ◽  
Cost Effective ◽  
Fever Virus ◽  
Economic Losses ◽  
Swine Industry ◽  
Virus Challenge

Abstract Background: Outbreaks of Classical swine fever virus (CSFV) cause significant economic losses in the swine industry. Vaccination is the major method to prevent and control the disease. As live attenuated vaccines fail to elicit differentiable immunity between infected and vaccinated animals, subunit vaccine was considered as an alternative candidate to prevent and eradicate CSFV. Subunit vaccines present advantages in DIVA immunogenicity and safety. The technology was limited due to the low yield and the high cost with multiple and large doses. The native E2 signal peptide has not been well defined before. Here, the aim of this study is to develop a cost-effective and efficacious E2 vaccine candidate against CSFV with signal peptide and E2 sequence selection. Results: A novel CSFV E2 sequence (E2ZJ) was identified from an epidemic strain of Zhejiang for outstanding secretion in baculovirus and enhanced immunogenicity. E2 secretion induced with the selected signal peptide, SPZJ (SP23), increase at least 50% as compared to any other signal peptides tested. Besides, unique antigenic features were identified in E2ZJ. E2ZJ elicited CSFV antibodies at the earlier stage than other E2 types tested in mice. Moreover, higher level of neutralization antibodies against both genotypes 1 and 2 CSFV with E2ZJ was detected than other E2s with the same dosage. Further, in piglets, E2ZJ successfully elicited neutralizing immunity. A single dose of 5 μg of E2ZJ was sufficient to induce protective antibodies against CSFV in piglets and provided 100% protection against lethal virus challenge. Conclusions: Our studies provide evidence that E2ZJ guided by a novel E2 signal peptide (SPZJ) was efficiently secreted and presented significantly improved immunogenicity than conventional E2 vaccines. Moreover, a single dose of 5 μg E2ZJ is efficacious against CSFV in piglets. Keywords: Classical swine fever virus; novel signal peptide; SPZJ-E2ZJ; subunit vaccine; protective immunity

scholarly journals Immunogenicity in Swine of Orally Administered Recombinant Lactobacillus plantarum Expressing Classical Swine Fever Virus E2 Protein in Conjunction with Thymosin α-1 as an Adjuvant

2015 ◽  
Vol 81 (11) ◽  
pp. 3745-3752 ◽  
Author(s):  
Yi-Gang Xu ◽  
Xue-Ting Guan ◽  
Zhong-Mei Liu ◽  
Chang-Yong Tian ◽  
Li-Chun Cui
Keyword(s):  
Immune Responses ◽  
Lactobacillus Plantarum ◽  
Classical Swine Fever Virus ◽  
Oral Vaccine ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
E2 Protein ◽  
Content Type ◽  
Cellular Immune

ABSTRACTClassical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious disease that results in enormous economic losses in pig industries. The E2 protein is one of the main structural proteins of CSFV and is capable of inducing CSFV-neutralizing antibodies and cytotoxic T lymphocyte (CTL) activitiesin vivo. Thymosin α-1 (Tα1), an immune-modifier peptide, plays a very important role in the cellular immune response. In this study, genetically engineeredLactobacillus plantarumbacteria expressing CSFV E2 protein alone (L. plantarum/pYG-E2) and in combination with Tα1 (L. plantarum/pYG-E2-Tα1) were developed, and the immunogenicity of each as an oral vaccine to induce protective immunity against CSFV in pigs was evaluated. The results showed that recombinantL. plantarum/pYG-E2 andL. plantarum/pYG-E2-Tα1 were both able to effectively induce protective immune responses in pigs against CSFV infection by eliciting immunoglobulin A (IgA)-based mucosal, immunoglobulin G (IgG)-based humoral, and CTL-based cellular immune responses via oral vaccination. Significant differences (P< 0.05) in the levels of immune responses were observed betweenL. plantarum/pYG-E2-Tα1 andL. plantarum/pYG-E2, suggesting a better immunogenicity ofL. plantarum/pYG-E2-Tα1 as a result of the Tα1 molecular adjuvant that can enhance immune responsiveness and augment specific lymphocyte functions. Our data suggest that the recombinantLactobacillusmicroecological agent expressing CSFV E2 protein combined with Tα1 as an adjuvant provides a promising strategy for vaccine development against CSFV.

Rapid and visual detection of Meloidogyne hapla using recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay

Plant Disease ◽  
2020 ◽  
Author(s):  
Zhiqiang Song ◽  
Xiai Yang ◽  
Xiaowei Zhang ◽  
Mingbao Luan ◽  
Bing Guo ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Meloidogyne Hapla ◽  
Root Knot Nematode ◽  
Resource Limited ◽  
Lateral Flow Dipstick ◽  
Wide Range ◽  
And Control

The northern root-knot nematode, Meloidogyne hapla, is a biotrophic parasite that infects many crops and causes severe economic losses worldwide. Rapid and accurate detection of M. hapla is crucial for disease forecasting and control. We developed a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay for rapid detection of M. hapla. The primers and a probe were designed based on the effector gene 16D10 sequence and were highly specific to M. hapla. The RPA reaction was performed at a wide range of temperatures from 25 to 45°C within 5 to 25 min, and the amplicon was visualized directly on the LFD within 5 min. The detection limits of the RPA-LFD assay were 10-3 female and 10-2 J2/0.5 g of soil, which was 10 times more sensitive than the conventional PCR assay. In addition, the RPA-LFD assay can detect M. hapla from infested plant roots and soil samples, and the entire detection process can be completed within 1.5 h. These results indicate that the RPA-LFD assay is a simple, rapid, specific, sensitive, and visual method that can be used for rapid detection of M. hapla in the field and in resource-limited conditions.

scholarly journals Crystal Structure of Classical Swine Fever Virus NS5B Reveals a Novel N-Terminal Domain

2018 ◽  
Vol 92 (14) ◽  
Author(s):  
Weiwei Li ◽  
Baixing Wu ◽  
Wibowo Adian Soca ◽  
Lei An
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Structural Basis ◽  
Economic Losses ◽  
Rna Dependent Rna Polymerase ◽  
Content Type ◽  
Terminal Domain

ABSTRACTClassical swine fever virus (CSFV) is the cause of classical swine fever (CSF). Nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is a key enzyme initiating viral RNA replication by ade novomechanism. It is also an attractive target for the development of anti-CSFV drugs. To gain a better understanding of the mechanism of CSFV RNA synthesis, here, we solved the first crystal structure of CSFV NS5B. Our studies show that the CSFV NS5B RdRp contains the characteristic finger, palm, and thumb domains, as well as a unique N-terminal domain (NTD) that has never been observed. Mutagenesis studies on NS5B validated the importance of the NTD in the catalytic activity of this novel RNA-dependent RNA polymerase. Moreover, our results shed light on CSFV infection.IMPORTANCEPigs are important domesticated animals. However, a highly contagious viral disease named classical swine fever (CSF) causes devastating economic losses. Classical swine fever virus (CSFV), the primary cause of CSF, is a positive-sense single-stranded RNA virus belonging to the genusPestivirus, familyFlaviviridae. Genome replication of CSFV depends on an RNA-dependent RNA polymerase (RdRp) known as NS5B. However, the structure of CSFV NS5B has never been reported, and the mechanism of CSFV replication is poorly understood. Here, we solve the first crystal structure of CSFV NS5B and analyze the functions of the characteristic finger, palm, and thumb domains. Additionally, our structure revealed the presence of a novel N-terminal domain (NTD). Biochemical studies demonstrated that the NTD of CSFV NS5B is very important for RdRp activity. Collectively, our studies provide a structural basis for future rational design of anti-CSFV drugs, which is critically important, as no effective anti-CSFV drugs have been developed.

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