Novel Application of Nanofluidic Chip Digital PCR for Detection of African Swine Fever Virus

African swine fever virus (ASFV) gives rise to a grievous transboundary and infectious disease, African swine fever (ASF), which has caused a great economic loss in the swine industry. To prevent and control ASF, once suspicious symptoms have presented, the movement of animal and pork products should be stopped, and then, laboratory testing should be adopted to diagnose ASF. A method for ASFV DNA quantification is presented in this research, which utilizes the next-generation PCR platform, nanofluidic chip digital PCR (cdPCR). The cdPCR detection showed good linearity and repeatability. The limit of detection for cdPCR is 30.1995 copies per reaction, whereas no non-specific amplification curve was found with other swine viruses. In the detection of 69 clinical samples, the cdPCR showed significant consistency [91.30% (63/69)] to the Office International des Epizooties-approved quantitative PCR. Compared with the commercial quantitative PCR kit, the sensitivity of the cdPCR assay was 86.27% (44/50), and the specificity was 94.44% (17/18). The positive coincidence rate of the cdPCR assay was 88% (44/50). The total coincidence rate of the cdPCR and kit was 89.86% (62/69), and the kappa value reached 0.800 (P < 0.0001). This is the first time that cdPCR has been applied to detecting ASFV successfully.

Download Full-text

BA71ΔCD2: a New Recombinant Live Attenuated African Swine Fever Virus with Cross-Protective Capabilities

Journal of Virology ◽

10.1128/jvi.01058-17 ◽

2017 ◽

Vol 91 (21) ◽

Cited By ~ 51

Author(s):

Paula L. Monteagudo ◽

Anna Lacasta ◽

Elisabeth López ◽

Laia Bosch ◽

Javier Collado ◽

...

Keyword(s):

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Sub Saharan Africa ◽

Swine Industry ◽

Lethal Challenge ◽

Major Threat ◽

Attenuated Viruses ◽

Genotype Ii

ABSTRACT African swine fever is a highly contagious viral disease of mandatory declaration to the World Organization for Animal Health (OIE). The lack of available vaccines makes its control difficult; thus, African swine fever virus (ASFV) represents a major threat to the swine industry. Inactivated vaccines do not confer solid protection against ASFV. Conversely, live attenuated viruses (LAV), either naturally isolated or obtained by genetic manipulation, have demonstrated reliable protection against homologous ASFV strains, although little or no protection has been demonstrated against heterologous viruses. Safety concerns are a major issue for the use of ASFV attenuated vaccine candidates and have hampered their implementation in the field so far. While trying to develop safer and efficient ASFV vaccines, we found that the deletion of the viral CD2v (EP402R) gene highly attenuated the virulent BA71 strain in vivo. Inoculation of pigs with the deletion mutant virus BA71ΔCD2 conferred protection not only against lethal challenge with the parental BA71 but also against the heterologous E75 (both genotype I strains). The protection induced was dose dependent, and the cross-protection observed in vivo correlated with the ability of BA71ΔCD2 to induce specific CD8+ T cells capable of recognizing both BA71 and E75 viruses in vitro. Interestingly, 100% of the pigs immunized with BA71ΔCD2 also survived lethal challenge with Georgia 2007/1, the genotype II strain of ASFV currently circulating in continental Europe. These results open new avenues to design ASFV cross-protective vaccines, essential to fight ASFV in areas where the virus is endemic and where multiple viruses are circulating. IMPORTANCE African swine fever virus (ASFV) remains enzootic in most countries of Sub-Saharan Africa, today representing a major threat for the development of their swine industry. The uncontrolled presence of ASFV has favored its periodic exportation to other countries, the last event being in Georgia in 2007. Since then, ASFV has spread toward neighboring countries, reaching the European Union's east border in 2014. The lack of available vaccines against ASFV makes its control difficult; so far, only live attenuated viruses have demonstrated solid protection against homologous experimental challenges, but they have failed at inducing solid cross-protective immunity against heterologous viruses. Here we describe a new LAV candidate with unique cross-protective abilities: BA71ΔCD2. Inoculation of BA71ΔCD2 protected pigs not only against experimental challenge with BA71, the virulent parental strain, but also against heterologous viruses, including Georgia 2007/1, the genotype II strain of ASFV currently circulating in Eastern Europe.

Download Full-text

A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus

Viruses ◽

10.3390/v11121129 ◽

2019 ◽

Vol 11 (12) ◽

pp. 1129 ◽

Cited By ~ 9

Author(s):

Ferenc Olasz ◽

István Mészáros ◽

Szilvia Marton ◽

Győző L. Kaján ◽

Vivien Tamás ◽

...

Keyword(s):

Sample Preparation ◽

African Swine Fever Virus ◽

Animal Health ◽

African Swine Fever ◽

Fever Virus ◽

Whole Genome ◽

Swine Industry ◽

Viral Dna ◽

Simple Method ◽

Generation Sequencing

In the recent years, African swine fever has become the biggest animal health threat to the swine industry. To facilitate quick genetic analysis of its causative agent, the African swine fever virus (ASFV), we developed a simple and efficient method for next generation sequencing of the viral DNA. Execution of the protocol does not demand complicated virus purification steps, enrichment of the virus by ultracentrifugation or of the viral DNA by ASFV-specific PCRs, and minimizes the use of Sanger sequencing. Efficient DNA-se treatment, monitoring of sample preparation by qPCR, and whole genome amplification are the key elements of the method. Through detailed description of sequencing of the first Hungarian ASFV isolate (ASFV_HU_2018), we specify the sensitive steps and supply key reference numbers to assist reproducibility and to facilitate the successful use of the method for other ASFV researchers.

Download Full-text

Estimation of the transmission dynamics of African swine fever virus within a swine house

Epidemiology and Infection ◽

10.1017/s0950268817001613 ◽

2017 ◽

Vol 145 (13) ◽

pp. 2787-2796 ◽

Cited By ~ 3

Author(s):

J. P. NIELSEN ◽

T. S. LARSEN ◽

T. HALASA ◽

L. E. CHRISTIANSEN

Keyword(s):

African Swine Fever Virus ◽

African Swine Fever ◽

Simulation Models ◽

Fever Virus ◽

Economic Consequences ◽

Estimation Of Parameters ◽

Swine Industry ◽

Transmission Parameters ◽

The Individual ◽

The Impact

SUMMARYThe spread of African swine fever virus (ASFV) threatens to reach further parts of Europe. In countries with a large swine production, an outbreak of ASF may result in devastating economic consequences for the swine industry. Simulation models can assist decision makers setting up contingency plans. This creates a need for estimation of parameters. This study presents a new analysis of a previously published study. A full likelihood framework is presented including the impact of model assumptions on the estimated transmission parameters. As animals were only tested every other day, an interpretation was introduced to cover the weighted infectiousness on unobserved days for the individual animals (WIU). Based on our model and the set of assumptions, the within- and between-pen transmission parameters were estimated to βw = 1·05 (95% CI 0·62–1·72), βb = 0·46 (95% CI 0·17–1·00), respectively, and the WIU = 1·00 (95% CI 0–1). Furthermore, we simulated the spread of ASFV within a pig house using a modified SEIR-model to establish the time from infection of one animal until ASFV is detected in the herd. Based on a chosen detection limit of 2·55% equivalent to 10 dead pigs out of 360, the disease would be detected 13–19 days after introduction.

Download Full-text

Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay during an Outbreak in Timor-Leste

Viruses ◽

10.3390/v12121444 ◽

2020 ◽

Vol 12 (12) ◽

pp. 1444

Author(s):

Peter T. Mee ◽

Shani Wong ◽

Kim J. O’Riley ◽

Felisiano da Conceição ◽

Joanita Bendita da Costa Jong ◽

...

Keyword(s):

African Swine Fever Virus ◽

African Swine Fever ◽

Lamp Assay ◽

Fever Virus ◽

Isothermal Amplification ◽

Clinical Samples ◽

Loop Mediated Isothermal Amplification ◽

Field Verification ◽

Extraction Step ◽

Timor Leste

Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.

Download Full-text

Identification of a Continuously Stable and Commercially Available Cell Line for the Identification of Infectious African Swine Fever Virus in Clinical Samples

Viruses ◽

10.3390/v12080820 ◽

2020 ◽

Vol 12 (8) ◽

pp. 820 ◽

Cited By ~ 2

Author(s):

Ayushi Rai ◽

Sarah Pruitt ◽

Elizabeth Ramirez-Medina ◽

Elizabeth A. Vuono ◽

Ediane Silva ◽

...

Keyword(s):

Cell Line ◽

Virus Isolation ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Clinical Samples ◽

Outbreak Strain ◽

Domestic Pigs ◽

Current Outbreak ◽

High Degree

African swine fever virus (ASFV) is causing outbreaks both in domestic pigs and wild boar in Europe and Asia. In 2018, the largest pig producing country, China, reported its first outbreak of African swine fever (ASF). Since then, the disease has quickly spread to all provinces in China and to other countries in southeast Asia, and most recently to India. Outbreaks of the disease occur in Europe as far west as Poland, and one isolated outbreak has been reported in Belgium. The current outbreak strain is highly contagious and can cause a high degree of lethality in domestic pigs, leading to widespread and costly losses to the industry. Currently, detection of infectious ASFV in field clinical samples requires accessibility to primary swine macrophage cultures, which are infrequently available in most regional veterinary diagnostic laboratories. Here, we report the identification of a commercially available cell line, MA-104, as a suitable substrate for virus isolation of African swine fever virus.

Download Full-text

Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus

PLoS ONE ◽

10.1371/journal.pone.0254815 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0254815

Author(s):

Jinyu Fu ◽

Yueping Zhang ◽

Guang Cai ◽

Geng Meng ◽

Shuobo Shi

Keyword(s):

African Swine Fever Virus ◽

Point Of Care ◽

African Swine Fever ◽

High Sensitivity ◽

Fluorescence Assay ◽

Fever Virus ◽

Diagnostic Methods ◽

High Morbidity ◽

Swine Industry ◽

Control And Prevention

African swine fever (ASF) is a serious contagious disease that causes fatal haemorrhagic fever in domestic and wild pigs, with high morbidity. It has caused devastating damage to the swine industry worldwide, necessitating the focus of attention on detection of the ASF pathogen, the African swine fever virus (ASFV). In order to overcome the disadvantages of conventional diagnostic methods (e.g. time-consuming, demanding and unintuitive), quick detection tools with higher sensitivity need to be explored. In this study, based on the conserved p72 gene sequence of ASFV, we combined the Cas12a-based assay with recombinase polymerase amplification (RPA) and a fluorophore-quencher (FQ)-labeled reporter assay for rapid and visible detection. Five crRNAs designed for Cas12a-based assay showed specificity with remarkable fluorescence intensity under visual inspection. Within 20 minutes, with an initial concentration of two copies of DNA, the assay can produce significant differences between experimental and negative groups, indicating the high sensitivity and rapidity of the method. Overall, the developed RPA-Cas12a-fluorescence assay provides a fast and visible tool for point-of-care ASFV detection with high sensitivity and specificity, which can be rapidly performed on-site under isothermal conditions, promising better control and prevention of ASF.

Download Full-text

Development of an Indirect ELISA to Detect African Swine Fever Virus pp62 Protein-Specific Antibodies

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.798559 ◽

2022 ◽

Vol 8 ◽

Author(s):

Kexin Zhong ◽

Mengmeng Zhu ◽

Qichao Yuan ◽

Zhibang Deng ◽

Simeng Feng ◽

...

Keyword(s):

Indirect Elisa ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Viral Disease ◽

Fever Virus ◽

Serum Samples ◽

Swine Industry ◽

Detection Techniques ◽

Positive Serum ◽

Great Reactivity

African swine fever (ASF) is a highly detrimental viral disease caused by African swine fever virus (ASFV). The occurrence and prevalence of this disease have become a serious threat to the global swine industry and national economies. At present, the detection volume of African swine fever is huge, more sensitive and accurate detection techniques are needed for the market. pp62 protein, as a protein in the late stage of infection, has strong antigenicity and a high corresponding antibody titer in infected pigs. In this study, the CP530R gene was cloned into expression vector pET-28a to construct a prokaryotic expression plasmid, which was induced by IPTG to express soluble pp62 protein. Western blot analysis showed that it had great reactivity. Using the purified recombinant protein as an antigen, an indirect ELISA method for detecting ASFV antibody was established. The method was specific only to ASFV-positive serum, 1:1600 diluted positive serum could still be detected, and the coefficients of variation (CV) of the intra assay and inter assay were both <10%. It turns out that the assays had excellent specificity, sensitivity, and repeatability. This provides an accurate, rapid, and economical method for the detection of ASFV antibody in clinical pig serum samples.

Download Full-text

African Swine Fever Virus Induces STAT1 and STAT2 Degradation to Counteract IFN-I Signaling

Frontiers in Microbiology ◽

10.3389/fmicb.2021.722952 ◽

2021 ◽

Vol 12 ◽

Author(s):

Elena Riera ◽

Daniel Pérez-Núñez ◽

Raquel García-Belmonte ◽

Lisa Miorin ◽

Adolfo García-Sastre ◽

...

Keyword(s):

Nuclear Translocation ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Swine Industry ◽

Inhibitory Mechanisms ◽

Host Innate Immune Response ◽

Serious Disease ◽

New Strategies ◽

Stat Pathway

African swine fever virus (ASFV) causes a serious disease in domestic pigs and wild boars and is currently expanding worldwide. No safe and efficacious vaccines against ASFV are available, which threats the swine industry worldwide. African swine fever virus (ASFV) is a complex dsDNA virus that displays multiple mechanisms to counteract the host innate immune response, whose efficacy might determine the different degrees of virulence displayed by attenuated and virulent ASFV strains. Here we report that infection with both virulent Arm/07/CBM/c2 and attenuated NH/P68 strains prevents interferon-stimulated gene (ISG) expression in interferon (IFN)-treated cells by counteracting the JAK/STAT pathway. This inhibition results in an impaired nuclear translocation of the interferon-stimulated gene factor 3 (ISGF3) complex, as well as in the proteasome-dependent STAT2 degradation and caspase 3-dependent STAT1 cleavage. The existence of two independent mechanisms of control of the JAK/STAT pathway, suggests the importance of preventing this pathway for successful viral replication. As ASFV virulence is likely associated with the efficacy of the IFN signaling inhibitory mechanisms, a better understanding of these IFN antagonistic properties may lead to new strategies to control this devastating pig disease.

Download Full-text

Subunit Vaccine Approaches for African Swine Fever Virus

Vaccines ◽

10.3390/vaccines7020056 ◽

2019 ◽

Vol 7 (2) ◽

pp. 56 ◽

Cited By ~ 13

Author(s):

Natasha N. Gaudreault ◽

Juergen A. Richt

Keyword(s):

Western Europe ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Swine Industry ◽

Fatal Disease ◽

The Caucasus ◽

Caucasus Region ◽

Highly Virulent ◽

Genotype Ii

African swine fever virus (ASFV) is the cause of a highly fatal disease in swine, for which there is no available vaccine. The disease is highly contagious and poses a serious threat to the swine industry worldwide. Since its introduction to the Caucasus region in 2007, a highly virulent, genotype II strain of ASFV has continued to circulate and spread into Eastern Europe and Russia, and most recently into Western Europe, China, and various countries of Southeast Asia. This review summarizes various ASFV vaccine strategies that have been investigated, with focus on antigen-, DNA-, and virus vector-based vaccines. Known ASFV antigens and the determinants of protection against ASFV versus immunopathological enhancement of infection and disease are also discussed.

Download Full-text