Evaluation of a Combined Live Attenuated Vaccine against Lumpy Skin Disease, Contagious Bovine Pleuropneumonia and Rift Valley Fever.

The use of effective vaccines is among the most important strategies for the prevention and progressive control of transboundary infectious animal diseases. However, the use of vaccine is often impeded by the cost, a lack of cold chains and other factors. In resource-limited countries in Africa, one approach to improve coverage and reduce cost is to vaccinate against multiple diseases using combined vaccines. Therefore, the objective of this study was to evaluate a combined vaccine for the prevention and control of Lumpy Skin Disease (LSD), Contagious Bovine Pleuropneumonia (CBPP) and Rift Valley fever (RVF). The LSD and CBPP were formulated as a combined vaccine, and the RVF was formulated separately as live attenuated vaccines. These consisted of a Mycoplasma MmmSC T1/44 strain that was propagated in Hayflick-modified medium, RVF virus vaccine, C13T strain prepared in African green monkey cells (Vero), and the LSDV Neethling vaccine strain prepared in primary testis cells. The vaccines were tested for safety via the subcutaneous route in both young calves and pregnant heifers with no side effect, abortion or teratogenicity. The vaccination of calves induced seroconversions for all three vaccines starting from day 7 post-vaccination (PV), with rates of 50% for LSD, 70% for CBPP and 100% for RVF, or rates similar to those obtained with monovalent vaccines. The challenge of cattle vaccinated with the LSD/CBPP and the RVF vaccine afforded full protection against virulent strains of LSDV and RVFV. A satisfactory level of protection against a CBPP challenge was observed, with 50% of protection at 6 months and 81% at 13 months PV. A mass vaccination trial was performed in four regions of Burkina Faso that confirmed safety and specific antibody responses induced by the vaccines. The multivalent LSD/CBPP+RVF vaccine provides a novel and beneficial approach to the control of the three diseases through one intervention and, therefore, reduces the cost and improves vaccination coverage.

A Survey of Henipavirus Tropism-Our Current Understanding from a Species/Organ and Cellular Level.

Henipaviruses are single-stranded RNA viruses that have been shown to be virulent in several species, including humans, pigs, horses, and rodents. Isolated nearly 30 years ago, these viruses have been shown to be of particular concern to public health, as at least two members (Nipah and Hendra viruses) are highly virulent, as well as zoonotic, and are thus classified as BSL4 pathogens. Although only 5 members of this genus have been isolated and characterized, metagenomics analysis using animal fluids and tissues has demonstrated the existence of other novel henipaviruses, suggesting a far greater degree of phylogenetic diversity than is currently known. Using a variety of molecular biology techniques, it has been shown that these viruses exhibit varying degrees of tropism on a species, organ/tissue, and cellular level. This review will attempt to provide a general overview of our current understanding of henipaviruses, with a particular emphasis on viral tropism.

Comparison of Gross Pathology between Classical and Recombinant Lumpy Skin Disease Viruses.

The pathology caused by three different isolates of lumpy skin disease virus, classical field cluster 1.2 strain Dagestan/2015, recombinant vaccine-like cluster 2.1 strain Saratov/2017, and cluster 2.2 strain Udmurtiya/2019, in cattle was compared from experimental infections. The infection of cattle was performed using intravenous administration of 2 mL of 105 TCID50/mL of each specific LSDV. Both classical and recombinant forms of LSDV cause pathological changes in the skin and lymph nodes, as well as the trachea and lungs. Due to circulatory disorders in the affected organs, multiple areas of tissue necrosis were observed, which, with the resurgence of secondary microflora, led to the development of purulent inflammation. Observed pathological changes caused by the recombinant vaccine-like strain Udmurtiya/2019 were characterized by a more pronounced manifestation of the pathoanatomical picture compared to the classical field strains Dagestan/2015 and Saratov/2017. Interestingly, Dagestan/2015 and Udmurtiya/2019 caused damage to the lymph nodes, characterized by serous inflammation and focal purulent lymphadenitis caused by purulent microflora. "Saratov/2017" did not cause pathology in the lymph nodes. All LSDVs were virulent and caused pathology, which was not distinguishable between viruses. This data set will serve as the experimentally validated basis for the comparative examination of novel LSDV strains in gross pathology.

Modular adjuvant-free pan-HLA-DR-immunotargeting subunit vaccine against SARS-CoV-2 elicits broad sarbecovirus-neutralizing antibody responses.

Subunit vaccines typically require co-administration with an adjuvant to elicit protective immunity, adding development hurdles that can impede rapid pandemic responses. To circumvent the need for adjuvant in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine, we engineer a thermostable immunotargeting vaccine (ITV) that leverages the pan-HLA-DR monoclonal antibody 44H10 to deliver the viral spike protein receptor-binding domain (RBD) to antigen-presenting cells. X-ray crystallography shows that 44H10 binds to a conserved epitope on HLA-DR, providing the basis for its broad HLA-DR reactivity. Adjuvant-free ITV immunization in rabbits and ferrets induces robust anti-RBD antibody responses that neutralize SARS-CoV-2 variants of concern and protect recipients from SARS-CoV-2 challenge. We demonstrate that the modular nature of the ITV scaffold with respect to helper T cell epitopes and diverse RBD antigens facilitates broad sarbecovirus neutralization. Our findings support anti-HLA-DR immunotargeting as an effective means to induce strong antibody responses to subunit antigens without requiring an adjuvant.

Development and laboratory evaluation of a competitive ELISA for serodiagnosis of Nipah and Hendra virus infection using recombinant Nipah glycoproteins and a monoclonal antibody.

Introduction: Nipah virus (NiV) and Hendra virus (HeV), of the genus Henipavirus, family Paramyxoviridae, are classified as Risk Group 4 (RG4) pathogens that cause respiratory disease in pigs and acute/febrile encephalitis in humans with high mortality. Methods: A competitive enzyme-linked immunosorbent assay (cELISA) using a monoclonal antibody (mAb) and recombinant NiV glycoprotein (G) was developed and laboratory evaluated using sera from experimental pigs, mini pigs and nonhuman primates. The test depends on competition between specific antibodies in positive sera and a virus-specific mAb for binding to NiV-G. Results: Based on 1,199 negative and 71 NiV positive serum test results, the cutoff value was determined as 35% inhibition. The diagnostic sensitivity and specificity of the NiV cELISA was 98.58 and 99.92%, respectively. When testing sera from animals experimentally infected with NiV Malaysia, the cELISA detected antibodies from 14 days post-infection (dpi) and remained positive until the end of the experiment (28 dpi). Comparisons using the Kappa coefficient showed strong agreement (100%) between the cELISA and a plaque reduction neutralization test (PRNT). Discussion: Because our cELISA is simpler, faster, and gives comparable or better results than PRNT, it would be an adequate screening test for suspect NiV and HeV cases, and it would also be useful for epidemiological surveillance of Henipavirus infections in different animal species without changing reagents.

Label-free impedimetric immunosensor for point-of-care detection of COVID-19 antibodies.

The COVID-19 pandemic has posed enormous challenges for existing diagnostic tools to detect and monitor pathogens. Therefore, there is a need to develop point-of-care (POC) devices to perform fast, accurate, and accessible diagnostic methods to detect infections and monitor immune responses. Devices most amenable to miniaturization and suitable for POC applications are biosensors based on electrochemical detection. We have developed an impedimetric immunosensor based on an interdigitated microelectrode array (IMA) to detect and monitor SARS-CoV-2 antibodies in human serum. Conjugation chemistry was applied to functionalize and covalently immobilize the spike protein (S-protein) of SARS-CoV-2 on the surface of the IMA to serve as the recognition layer and specifically bind anti-spike antibodies. Antibodies bound to the S-proteins in the recognition layer result in an increase in capacitance and a consequent change in the impedance of the system. The impedimetric immunosensor is label-free and uses non-Faradaic impedance with low nonperturbing AC voltage for detection. The sensitivity of a capacitive immunosensor can be enhanced by simply tuning the ionic strength of the sample solution. The device exhibits an LOD of 0.4 BAU/ml, as determined from the standard curve using WHO IS for anti-SARS-CoV-2 immunoglobulins; this LOD is similar to the corresponding LODs reported for all validated and established commercial assays, which range from 0.41 to 4.81 BAU/ml. The proof-of-concept biosensor has been demonstrated to detect anti-spike antibodies in sera from patients infected with COVID-19 within 1 h. Photolithographically microfabricated interdigitated microelectrode array sensor chips & label-free impedimetric detection of COVID-19 antibody.

Cell-Free Dot Blot: an Ultra-Low-Cost and Practical Immunoassay Platform for Detection of Anti-SARS-CoV-2 Antibodies in Human and Animal Sera.

Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused severe disruption to key aspects of human life globally and highlighted the need for timely, adaptive, and accessible pandemic response strategies. Here, we introduce the cell-free dot blot (CFDB) method, a practical and ultra-low-cost immune diagnostic platform capable of rapid response and mass immunity screening for the current and future pandemics. Similar in mechanism to the widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel and advantageous in that (i) it uses linear DNA to produce the target viral antigen fused to a SpyTag peptide in a cell-free expression system without the need for traditional cloning and antigen purification, (ii) it uses SpyCatcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as a universal secondary detection reagent, obviating the need for commercial or sophisticated enzyme conjugates, and (iii) sera are spotted directly on a nitrocellulose membrane, enabling a simple "dipping" mechanism for downstream incubation and washing steps, as opposed to individual processing of wells in a multiwell plate. To demonstrate the utility of our method, we performed CFDB to detect anti-severe acute respiratory syndrome coronavirus 2 nucleocapsid protein antibodies in precharacterized human sera (23 negative and 36 positive for COVID-19) and hamster sera (16 negative and 36 positive for COVID-19), including independent testing at a collaborating laboratory, and we show assay performance comparable to that of conventional ELISAs. At a similar capacity to 96-well plate ELISA kits, one CFDB assay costs only ~$3 USD. We believe that CFDB can become a valuable pandemic response tool for adaptive and accessible sero-surveillance in human and animal populations. IMPORTANCE The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method. Based on the principles of cell-free synthetic biology and alternative dot blotting procedures, our CFDB immunoassay is designed to provide for timely, practical, and low-cost responses to existing and emerging public health threats, such as the COVID-19 pandemic, at a similar throughput and comparable performance as conventional ELISAs. Notably, the molecular detection reagents used in CFDB can be produced rapidly in-house, using established protocols and basic laboratory infrastructure, minimizing reliance on strained commercial reagents. In addition, the materials and imaging instruments required for CFDB are the same as those used for common Western blotting experiments, further expanding the reach of CFDB in decentralized facilities.

Faradaic Impedimetric Immunosensor for Label-Free Point-of-Care Detection of COVID-19 Antibodies Using Gold-Interdigitated Electrode Array.

Label-free electrochemical biosensors have many desirable characteristics in terms of miniaturization, scalability, digitization, and other attributes associated with point-of-care (POC) applications. In the era of COVID-19 and pandemic preparedness, further development of such biosensors will be immensely beneficial for rapid testing and disease management. Label-free electrochemical biosensors often employ [Fe(CN)6]-3/4 redox probes to detect low-concentration target analytes as they dramatically enhance sensitivity. However, such Faradaic-based sensors are reported to experience baseline signal drift, which compromises the performance of these devices. Here, we describe the use of a mecaptohexanoic (MHA) self-assembled monolayer (SAM) modified Au-interdigitated electrode arrays (IDA) to investigate the origin of the baseline signal drift, developed a protocol to resolve the issue, and presented insights into the underlying mechanism on the working of label-free electrochemical biosensors. Using this protocol, we demonstrate the application of MHA SAM-modified Au-IDA for POC analysis of human serum samples. We describe the use of a label-free electrochemical biosensor based on covalently conjugated SARS-CoV-2 spike protein for POC detection of COVID-19 antibodies. The test requires a short incubation time (10 min), and has a sensitivity of 35.4/decade (35.4%/10 ng mL-1) and LOD of 21 ng/mL. Negligible cross reactivity to seasonal human coronavirus or other endogenous antibodies was observed. Our studies also show that Faradaic biosensors are ~17 times more sensitive than non-Faradaic biosensors. We believe the work presented here contributes to the fundamental understanding of the underlying mechanisms of baseline signal drift and will be applicable to future development of electrochemical biosensors for POC applications.

Capripoxviruses, leporipoxviruses, and orthopoxviruses: Occurrences of recombination.

Poxviruses are double-stranded DNA viruses with several members displaying restricted host ranges. They are genetically stable with low nucleotide mutation rates compared to other viruses, due to the poxviral high-fidelity DNA polymerase. Despite the low accumulation of mutations per replication cycle, poxvirus genomes can recombine with each other to generate genetically rearranged viruses through recombination, a process directly associated with replication and the aforementioned DNA polymerase. Orthopoxvirus replication is intimately tethered to high frequencies of homologous recombination between co-infecting viruses, duplicated sequences of the same virus, and plasmid DNA transfected into poxvirus-infected cells. Unfortunately, the effect of these genomic alterations on the cellular context for all poxviruses across the family Poxviridae remains elusive. However, emerging sequence data on currently circulating and archived poxviruses, such as the genera orthopoxviruses and capripoxviruses, display a wide degree of divergence. This genetic variability cannot be explained by clonality or genetic drift alone, but are probably a result of significant genomic alterations, such as homologous recombination, gene loss and gain, or gene duplications as the major selection forces acting on viral progeny. The objective of this review is to cross-sectionally overview the currently available findings on natural and laboratory observations of recombination in orthopoxviruses, capripoxviruses, and leporipoxviruses, as well as the possible mechanisms involved. Overall, the reviewed available evidence allows us to conclude that the current state of knowledge is limited in terms of the relevance of genetic variations across even a genus of poxviruses as well as fundamental features governing and precipitating intrinsic gene flow and recombination events.

Detection of Nipah and Hendra Viruses Using Recombinant Human Ephrin B2 Capture Virus in Immunoassays.

Nipah virus (NiV) and Hendra virus (HeV) are classified as high-consequence zoonotic viruses characterized by high pathogenicity and high mortality in animals and humans. Rapid diagnosis is essential to containing the outbreak. In this study, the henipavirus receptor ephrin B2 was examined to determine whether it could be used as a universal ligand for henipavirus detection in immunoassays. Enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant ephrin B2 as the capture ligand and two monoclonal antibodies (mAbs) as detection reagents. Using mAb F27NiV-34, which cross-reacts with NiV and HeV, we were able to detect NiV and HeV, while mAb F20NiV-65 was used to detect NiV. Therefore, using these two ELISAs, we were able to differentiate between NiV and HeV. Furthermore, we developed a rapid lateral flow strip test for NiV detection using ephrin B2 as the capture ligand combined with mAb F20NiV-65 as the detector. Taken together, our results show that the combination of ephrin B2 and a specific mAb provides an excellent pairing for NiV and HeV detection.

A gold nanoparticle-protein G electrochemical affinity biosensor for the detection of SARS-CoV-2 antibodies: a surface modification approach.

As COVID-19 waves continue to spread worldwide, demand for a portable, inexpensive and convenient biosensor to determine community immune/infection status is increasing. Here we describe an impedance-based affinity biosensor using Interdigitated Electrode (IDE) arrays to detect antibodies to SARS-CoV-2 in serum. We created the biosensor by functionalizing the IDEs' surface with abaculaovirus-expressed and purified Spike (S) protein to bind anti-SARS CoV-2antibodies. Gold nanoparticles (GNP) fused to protein G were used to probe for bound antibodies. An ELISA assay using horseradish peroxidase-protein G to probe for bound IgG confirmed that the purified S protein bound a commercial source of anti-SARS-CoV-2 antibodies specifically and bound anti-SARS-CoV-2 antibodies in COVID-19 positive serum. Then we demonstrated that our biosensor could detect anti-SARS-CoV-2 antibodies with 72% sensitivity in 2 h. Using GNP-protein G, the affinity biosensor had increased impedance changes with COVID-19positive serum and minimal or decreased impedance changes with negative serum. This demonstrated that our biosensor could discriminate between COVID-19 positive and negative sera, which were further improved using poly(vinyl alcohol)as a blocking agent.

Molecular and histopathological characterization of lumpy skin disease in cattle in northern Vietnam during the 2020-2021 outbreaks.

Lumpy skin disease (LSD) is a serious emerging infectious disease in cattle caused by a virus of the family Poxviridae. According to the Department of Animal Health, LSD first occurred in Vietnam at the end of October 2020 in Cao Bang and Lang Son provinces. So far, the disease has infected over 63,000 animals, resulting in 9170 deaths occurring in 32 different provinces in northern and central Vietnam. In this study, skin samples from lumpy skin disease virus (LSDV)-infected cattle from the northern provinces of Vietnam displaying clinical symptoms including fever (> 40 °C), runny nose, drooling, and skin lesions were used for genetic characterization and histopathology. Genetic analysis of the partial P32 (LSDV074), partial F (LSDV117), complete RPO30 (LSDV035), and complete G-protein-coupled-chemokine-like receptor (GPCR) (LSDV011) genes showed that all Vietnamese LSDV strains belonged to the genus Capripoxvirus and were closely related to LSDV strains isolated in China. Microscopic examination of the skin lesions showed thickening of the epidermal layer of the skin and hair follicles, hyperplasia of sebaceous glands, intracytoplasmic inclusion bodies, and hemorrhages in the mesoderm.

Improving immunoassay detection accuracy of anti-SARS-CoV-2 antibodies through dual modality validation.

A novel test strategy is proposed with dual-modality detection techniques for COVID-19 antibody detection. The full-length S protein of SARS-CoV-2 was chemically immobilized on a glass surface to capture anti-SARS-CoV-2 IgG in patient serum and was detected through either Electrochemical Impedance Spectroscopy (EIS) or fluorescence imaging with labeled secondary antibodies. Gold nanoparticles conjugated with protein G were used as the probe and the bound GNP-G was detected through EIS measurements. Anti-human-IgG conjugated with the fluorescent tag Alexa Fluor 488 was used as the probe for fluorescence imaging. Clinical SARS-CoV-2 IgG positive serum and negative controls were used to validate both modalities. For fluorescence-based detection, a high sensitivity was noticed with a quantification range of 0.01-0.1 A.U.C. and a LOD of 0.004 A.U.C. This study demonstrates the possibility of utilizing different measurement techniques in conjunction for improved COVID-19 serology testing.

An impedimetric biosensor for COVID-19 serology test and modification of sensor performance via dielectrophoresis force.

Coronavirus disease 2019 (COVID-19) has caused significant global morbidity and mortality. The serology test that detects antibodies against the disease causative agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has often neglected value in supporting immunization policies and therapeutic decision-making. The ELISA-based antibody test is time-consuming and bulky. This work described a gold micro-interdigitated electrodes (IDE) biosensor for COVID antibody detection based on Electrochemical Impedance Spectroscopy (EIS) responses. The IDE architecture allows easy surface modification with the viral structure protein, Spike (S) protein, in the gap of the electrode digits to selectively capture anti-S antibodies in buffer solutions or human sera. Two strategies were employed to resolve the low sensitivity issue of non-faradic impedimetric sensors and the sensor fouling phenomenon when using the serum. One uses secondary antibody-gold nanoparticle (AuNP) conjugates to further distinguish anti-S antibodies from the non-specific binding and obtain a more significant impedance change. The second strategy consists of increasing the concentration of target antibodies in the gap of IDEs by inducing an AC electrokinetic effect such as dielectrophoresis (DEP). AuNP and DEP methods reached a limit of detection of 200 ng/mL and 2 µg/mL, respectively using purified antibodies in buffer, while the DEP method achieved a faster testing time of only 30 min. Both strategies could qualitatively distinguish COVID-19 antibody-positive and -negative sera. Our work, especially the impedimetric detection of COVID-19 antibodies under the assistance of the DEP force presents a promising path toward rapid, point-of-care solutions for COVID-19 serology tests.

The changing epidemiology of lumpy skin disease in Russia since the first introduction from 2015 to 2020.

Lumpy skin disease (LSD) is an economically important transboundary disease affecting cattle, causing large economic losses such as decreased production and trade restrictions. LSD has been a historically neglected disease since it previously caused disease limited to the African continent. Currently, the epidemiology of LSD virus is based on how the disease is transmitted in tropical and sub-tropical climates. The understanding of its epidemiology in hemiboreal climates is not well understood and needs urgent attention to expand the current knowledge. In this study, the epidemiological findings on LSD in Russia over a 6-year period are summarized and discussed. A total of 471 outbreaks were identified spanning over a 9000 km range. The outbreaks of LSD occur primarily in small holder farms (backyard) compared to commercial farms between mid-May through mid-November including weather conditions with snow and freezing temperatures that preclude vector activity. Mortality and morbidity varied across the 6 years ranging from 1.19% to 61.8% and 0% to 50%, respectively, with a tendency to decline from 2015 to 2020. The geographic pattern of spread was assessed by means of directionality, indicating a northward movement from 2015 to 2016, with a consequent East turn in 2017 through Siberia to the Far East by 2020. All cases occurred along the border with Kazakhstan. Mathematical modelling showed that the disease tended to form statistically verified annual spatiotemporal clusters in 2016-2018, whereas in 2019 and 2020 such segregation was not evident. The trend of spread was mainly either from south to north or from south to a north-east direction.

Cloning Strategies for the Generation of Recombinant Capripoxvirus Through the Use of Screening and Selection Markers.

The ability to manipulate capripoxvirus through gene knockouts and gene insertions has become an increasingly valuable research tool in elucidating the function of individual genes of capripoxvirus, as well as in the development of capripoxvirus-based recombinant vaccines. The homologous recombination technique is commonly used to generate capripoxvirus knockout viruses (KO), and is based on the targeting of a particular viral gene of interest. This technique can also be used to insert a gene of interest. A protocol for the generation of a viral gene knockout is described. This technique involves the use of a plasmid which encodes the flanking sequences of the regions where the homologous recombination will occur, and will result in the insertion of an EGFP reporter gene for visualization of recombinant virus, as well as the E. coli gpt gene as a positive selection marker. If an additional gene is to be incorporated, this can be achieved by inserting a gene of interest for expression under a poxvirus promoter into the plasmid between the flanking regions for insertion. This chapter describes a protocol for generating such recombinant capripoxviruses. An alternative step for the removal of both the EGFP and gpt cassettes and an optional selection step using CRISPR technology are also described.

Development of multiplex real-time PCR assays for differential detection of capripoxvirus, parapoxvirus and foot-and-mouth disease virus.

This study reports the development of multiplex real-time PCR assays for differential detection of capripoxvirus (CaPV), parapoxvirus (PaPV) and foot-and-mouth disease virus (FMDV) in sheep, goats and cattle. Three multiplex assays were developed, a capripox (CaP) rule-out assay for simultaneous detection and differentiation of CaPV and PaPV, a FMD rule-out assay for simultaneous detection and differentiation of FMDV and PaPV, and a FMD/CaP rule-out assay for simultaneous detection and differentiation of CaPV, PaPV and FMDV. All multiplex assays included ß-actin gene ACTB as an internal positive control to monitor PCR inhibition and accuracy of nucleic acid extractions. The optimized assays were highly specific to the target viruses (CaPV, PaPV and FMDV) with no cross-reactivity against other viruses that cause similar clinical signs. Using positive control plasmids as template, the limit of detection (LOD) of the multiplex assays were estimated as 2 CaPV, 7 PaPV and 15 FMDV copies per assay. The amplification efficiency (AE) and correlation coefficient (R2 ), estimated from the standard curves (Ct vs. log10 template dilution), were 94%-106% and >0.99, respectively, for CaP and FMD rule-out assays, 96%-116% (AE) and >0.98 (R2 ), respectively, for CaP/FMD rule-out assays and 91%-102% and >0.99, respectively, for the corresponding singleplex assays. The diagnostic sensitivity (DSe) of the multiplex assays was assessed on 35 CaPV and 39 FMDV clinical specimens from experimentally infected (CS-E) animals, and 29 CaPV (LSDV), 28 FMDV and 36 PaPV clinical specimens from naturally infected (CS-N) animals; all tested positive (DSe 100%) except two CS-E FMDV specimens that were tested negative by FMD rule-out and the corresponding singleplex (FMDV) assays (37/39; DSe 95%). The newly developed multiplex assays offer a valuable tool for differential detection of clinically indistinguishable CaPV, PaPV and FMDV in suspected animals and animals with mixed infections.

Integrating Reverse Transcription Recombinase Polymerase Amplification with CRISPR Technology for the One-Tube Assay of RNA.

CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.

Development of a novel real-time PCR assay targeting p54 gene for rapid detection of African swine fever virus (ASFV) strains circulating in Vietnam.

African swine fever (ASF) continues to cause outbreaks throughout regions of Africa, Europe and Asia. The disease can cause severe morbidity and mortality resulting in serious economic losses. Since there is no vaccine available to control ASF, early detection is critical to contain and control the disease. The aim of this study was to develop a novel real-time PCR assay based on highly conserved ASFV gene E183L (p54). The limit of detection of the assay, VNUA-p54 real-time PCR, was 2.63 copies/reaction and 2 Log10 HAD50 /ml. The VNUA-p54 real-time PCR was able to detect fifteen different ASFV reference strains representing p72 genotypes I, II and V. The assay was specific and did not amplify other swine viruses including CSFV, FMDV, PRRSV and PEDV. The diagnostic sensitivity of the real-time PCR assay was evaluated using 200 field clinical specimens collected from swine farms located in different provinces in Vietnam. The VNUA-p54 real-time PCR assay is an additional tool for ASF diagnostics and can be used in combination with other p72 based ASFV real-time PCR assays as a rapid confirmatory assay.