Abstract:

African horse sickness (AHS) is an acute and fatal vector-borne infectious disease of equids, caused by the African horse sickness virus (AHSV).  The World Organization for Animal Health (WOAH) has classified AHS as a notifiable animal disease, and AHS has also been classified as a Class I animal infectious disease in China.  AHS is mainly found in Africa, the Middle East and the Arabian Peninsula.  China is currently recognized by the WOAH as an AHS-free zone.  However, in 2020, there were outbreaks of AHS in 2 countries neighboring China, Thailand and Malaysia (Bunpapong et al. 2021), which increases the risk of the introduction of AHS into China.  Therefore, in order to prevent the occurrence of AHS in China and to further monitor the spread of the disease, the development of rapid, accurate and cost-effective diagnostic methods for the detection of AHSV is essential.  

AHSV is a segmented double-stranded RNA virus belonging to the genus Orbivirus in the family Reoviridae.  It is mainly transmitted by midges (Maurer et al. 2022), and is able to infect all members of the Equidae, including horses, mules, donkeys, and zebras.  AHSV infection causes severe morbidity and mortality (up to 90%) in horses, while mules, donkeys and zebras are less susceptible than horses to the disease (Barnard 1998).  

The AHSV genome contains 10 double-stranded RNA segments, encoding 7 structural proteins (VP1–7) and 4 non-structural proteins (NS1–4).  AHSV is a complex non-enveloped virus with an icosahedral capsid comprising 3 distinct concentric protein layers.  VP2 and VP5 are the components of the outer capsid of the virion.  VP2 is the major determinant of AHSV serotype, and 9 serotypes (AHSV-1 to AHSV-9) have been identified according to the VP2 antigenicity; VP3 and VP7 are the components of the major inner capsid of the virion; VP1, VP4 and VP6 constitute the minor inner capsid of AHSV.  

AHSV RNA segment 7 (vp7) is highly conserved among all AHSV serotypes and is the primary molecular diagnostic target of AHSV.  The VP7 protein encoded by this segment is the major antigen of AHSV and is commonly used as a serological diagnostic for AHSV.  Real-time RT-PCR targeting vp7 is capable of detecting all known types of AHSV and is recommended by the WOAH for the detection of this virus (Aguero et al. 2008; Guthrie et al. 2013; WOAH 2019).  

Recently, molecular diagnostics for infectious diseases have been developed based on clustered regularly interspaced short palindromic repeats-associated Cas endonucleases (CRISPR/Cas) systems combined with isothermal amplification techniques (Chen J S et al. 2018; Gootenberg et al. 2018; Myhrvold et al. 2018).  Some Cas proteins with non-specific endonuclease activity, such as Cas12a, activate auxiliary (non-specific) cleavage of nearby single-stranded non-target nucleic acids upon recognition of the target.  By modifying a single-stranded nucleic acid with a fluorophore quencher, which fluoresces upon cleavage of the Cas12a and crRNA complex, this activity can be used to detect the presence of specific cleavage.  The CRISPR/Cas12-based detection system has certain advantages over traditional nucleic acid diagnostic methods (qPCR), including rapidity, simplicity, low cost, and low equipment requirements.  In this study, we developed a sensitive detection method for AHSV using the CRISPR/Cas12a system combined with reverse transcription-recombinase-assisted amplification (RT-RAA) (CRISPR/Cas12a-RT-RAA), which specifically targets the vp7 RNA of AHSV.  

To generate a CRISPR/Cas12-based AHSV detection system, a Cas12a from the Lachnospiraceae bacterium, LbCas12a protein, was first expressed in an Escherichia coli system and purified with Strep-Tactin Sepharose resin (Appendix A).  A single-stranded DNA (ssDNA) reporter labeled with a fluorophore and a quencher at the 2 termini (5´-6-FAM-TTATT-BHQ-3´) was synthesized by Sangon Biotech (Shanghai, China).  Ten crRNAs were designed to target the conserved region of the vp7 sequence of all AHSV strains (Appendices B and C).  These crRNAs with a repeat sequence were prepared using in vitro transcription following a previously described method (Wang et al. 2023).  In order to screen for an optimal crRNA for the sensitive detection of AHSV, 10 crRNAs were individually tested using a 25 μL CRISPR/Cas12-based reaction volume containing 0.4 μmol L^–1 LbCas12a, 0.4 μmol L^–1 ssDNA reporter, 1.2 μmol L^–1 crRNA, 10⁹ copies μL^–1 vp7 plasmid DNA (pMD18-T-vp7, containing the entire vp7 sequence) and 2.5 μL NEBuffer 2.1 (10×).  The reaction was performed at 37°C for 50 min on a qPCR thermal cycler (Applied Biosystems QuantStusio 5 Real-Time PCR System, USA) with fluorescence measurements taken every 30 s.  Fluorescence detection suggested that crRNA10 showed the highest efficiency in this reaction system (Fig. 1-A).  Therefore, crRNA10 was identified as the best option for the AHSV CRISPR/Cas12a detection platform and was used in the subsequent experiments.

Recombinase-assisted amplification (RAA) is an isothermal amplification technique that has been widely used to detect microbial pathogens (Chen C et al. 2018; Wang et al. 2020; Xue et al. 2020).  Recent studies have increasingly integrated the RAA assay with the CRISPR-Cas system, which provides a second detection step for amplification products, increasing detection sensitivity and specificity, and enabling more convenient and intuitive determination of detection results (Li et al. 2023).  Six specific RAA primers specifically targeting vp7 were designed based on the flanking sequence of the crRNA10 region (Appendix D).  To screen for the optimal RAA primer pair for the sensitive detection of AHSV, a standard RAA reaction was performed with pMD18-T-vp7 (at a concentration of 10⁹ copies μL^–1) as a template, using the RAA Nucleic Acid Amplification Kit (Qitian, China) and following the manufacturer’s instructions.  Following RAA amplification at 37°C for 30 min, the products of the RAA amplification were used as substrates for the CRISPR/Cas12a system detection.  As shown in Fig. 1-B, the strongest fluorescence signals for the CRISPR/Cas12a-RAA assay were detected when the F3/R3 primer pair was used.  The results showed that the F3/R3 primer pair had the best amplification efficiency and was therefore selected for the establishment of the CRISPR/Cas12a-RAA detection platform and used for subsequent experiments.

To evaluate the sensitivity of the CRISPR/Cas12a-RAA detection platform, we prepared vp7 RNA in vitro using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, USA), using vp7 PCR products and with T7 promoter sequences as templates.  A total of 1 μL of vp7 RNA at different concentrations was used as a template for the RT-RAA reaction using RT-RAA Nucleic Acid Amplification Kit (Qitian, Wuxi, China) for 30 min, and then 1 μL of RAA amplification product was extracted and used as a substrate for the CRISPR/Cas12a detection system and reacted for 30 min.  The CRISPR/Cas12a-RT-RAA assay was developed in this way.  As shown in Fig. 1-C, the detection limit of the CRISPR/Cas12a-RT-RAA assay was 10 copies of the vp7 mRNA molecule per reaction.  However, the detection limit of the real-time RT-PCR assay established by Aguero in 2008 was 100 copies of the vp7 RNA molecule per reaction (Aguero et al. 2008) (Fig. 1-D).  In 2015, WOAH organized the AHS reference laboratory to conduct a comparison of trials to evaluate different conventional detection methods, and confirmed that the real-time RT-PCR established by Aguero in 2008 was one of the best detection methods for diagnosing AHSV (WOAH 2019).  Our results suggest that the CRISPR/Cas12a-RT-RAA assay has higher sensitivity compared to the real-time RT-PCR assay.

To test the specificity of the CRISPR/Cas12a-RT-RAA assay to AHSV, other equine viral and bacterial pathogens were tested, including equine infectious anemia virus (EIAV), equine influenza virus (EIV), equine arthritis virus (EAV), equine herpesvirus-1 (EHV-1), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi), and Salmonella enterica subsp. enterica serovar Abortusequi (S. Abortusequi).  All of these pathogens were stored in our laboratory and RNA/DNA from these pathogens was prepared as previously described (Chen et al. 2022).  As shown in Fig. 1-E, no fluorescence was observed when these equine pathogens were tested using the CRISPR/Cas12a-RT-RAA assay, whereas significant fluorescence was observed when the vp7 mRNA was tested, indicating that this CRISPR/Cas12a-RT-RAA method is highly specific for the detection of AHSV.

Due to the lack of AHSV-positive samples, we evaluated the performance of the CRISPR/Cas12a-RT-RAA assay in clinical practice by adding vp7 mRNAs to mRNAs extracted from equine blood or tissue samples as positive controls, and equine blood or equine tissue mRNAs without vp7 mRNAs as negative controls.  A total of 20 equine mRNA samples, including 5 equine blood cell mRNA samples (S1–5), 5 equine blood cell mRNA samples with vp7 mRNA (S6–10), 5 equine tissue (heart, liver, spleen, lung and kidney) mRNA (S11–15), and 5 equine tissue mRNA with vp7 mRNA (S16–20), were prepared and assessed using the CRISPR/Cas12a-RT-RAA assay.  As shown in Fig. 1-F, strong fluorescence signals were detected in all equine mRNA samples with vp7 mRNA, but not in any equine mRNA samples without vp7 mRNA.  This result demonstrates that the CRISPR/Cas12a-RT-RAA assay is able to detect vp7 mRNA efficiently in samples collected under complex conditions and can be used as a back-up technology for the early field detection of AHSV.

In conclusion, we reported the development and validation of a CRISPR/Cas12a-combined RT-RAA-based detection assay for AHSV with high specificity, sensitivity and convenience.  This assay targets the vp7 mRNA, a highly conserved segment of the AHSV genome that has not been observed to cross-react with the nucleic acids of 7 common equine pathogens, including EIAV, EIV, EAV, EHV-1, EHV-4, S. equi, and S. Abortusequi.  Notably, this assay was 10 times more sensitive than real-time RT-PCR.  In addition, the signal generated by the assay would be directly visible to the naked eye under UV light without the need for special instrumentation.  Therefore, the CRISPR/Cas12a combination RT-RAA assay developed here has the potential to be used as an alternative to traditional real-time RT-PCR assays for the rapid diagnosis of AHSV infection.  We will continue to optimize and improve the assay and expect that it will allow the detection of AHSV in the field and improve the early warning and diagnosis of AHS.