Before 2016, CSF in China was under controlled with the effort of the compulsory vaccination policy of C strain vaccine [16, 17]. However, with the development of intensive farming, many large-scale pig farms have been built in China and the compulsory vaccination policy has been replaced by overall vaccination since July 2016, the epidemiology of CSFV in turn has changed significantly [18]. Chronic and atypical CSF become dominant in epidemic outbreaks, which characterized as sporadic, vulnerable in young age, persistent infection, complex onset and immune tolerance, which have brought new challenges to the prevention and control of CSF [19, 20]. In particular, it is clinically difficult to distinguish between infection and vaccination, which brings new challenges to the prevention, control and elimination of CSFV in China. The outbreaks of ASF in 2018 in China has devastating impact on the country's pig industry [21]. With the rapid response of our government and a series of precise control policies, the outbreak has been effectively controlled and the pig industry has been recovering in an orderly way [22]. In 2021, however, new situations for ASF epidemic in China have emerged, which showed reduced mortality, atypical clinical signs, and some "natural variant strains" showed no hemadsorption (HAD) [23]. These natural variant ASFV strains caused subclinical symptoms, which were difficult to identify and detect at the early stage and can be easily confused with other diseases, leading to problems in differential diagnosis and prevention and control of ASFV [24]. APPV, commonly known as "piglet shivering disease" or "jumping disease", is a disease in which piglets’ exhibit paroxysmal muscle movements in the head, limbs and other parts of the body [25]. It can cause piglets difficulties in standing, blocked suckling and even death. It is estimated that the number of piglets weaned by APPV infected sows could reduce by 10%, and the mortality rate of newborn piglets affected by APPV could rise up to 30% due to malnutrition [26]. APPV is widespread in pig herds throughout China and the world, posing a serious threat to pig industry [27]. CSFV, ASFV and APPV have become three important contagious virulent infectious diseases in China, which show similar clinical signs and are difficult to distinguish from each other. It is, therefore, critical to develop a simple, rapid, specific and sensitive assay to differential diagnose these three diseases.
The sensitivity of the gene chip assay developed in this study is 6.98 copies/µL and 69.2 copies/µL for CSFV-W and CSFV-V respectively, which is higher than some published CSFV RT-PCR assays, demonstrating the advantage of the gene chip assay in sensitivity. In the current study, after looking through the 5’UTR gene sequences of 30 CSFV field strains preserved in our laboratory and 10 published strains on NCBI and 4 vaccine strains, we found that the 5'UTR was highly conserved among them and many CSFV RT-PCR diagnostic methods also chose this region for primer design [28, 29]. NS5B is an RNA polymerase involved in viral genome replication and is one of the popular target for CSFV genotyping. We found there was a base difference between the vaccine and wild strains in the NS5B region which was then used for MGB probe designed to specifically binding to the NS5B gene of CSFV vaccine strains. It is not only practical to C strain, which is widely used in China, but also applicable to the CSFV low-temperature mutagenesis vaccine (Thiveosal strain).
The biological reaction between samples and the gene chip is critical for the success detection and subsequent analysis of the gene chip assay. The size of the gene probe and the length of PCR products on the microarray are also important factors affecting the hybridization signal of the microarray [30]. Therefore, the length of the probes designed in this experiment is less than 30 bp, and the length of the PCR products is less than 100 bp, thus ensuring a stable and clear signal response. To facilitate the interpretation of the microarray results, primers were labelled with biotin that has good affinity with streptavidin. After quadruple PCR amplification, the products were combined with the probe on the microarray and reacted with the HRP-labelled streptavidin. Conventional gene microarrays usually use aldehyde-based slides as support and take longer time for detection. This assay use "0 + X" nano-membranes (0 for zero background and X for various probes) supported by high-topping materials which significantly reduces the reaction time compared to conventional one, resulting in significant time and cost savings. The hybridization process is simple and time-consuming. The results are fully consistent with those of national standard assays. The assay also allows for the addition of other swine infectious diseases other than the three diseases we target in this study. Therefore, the gene chip assay has potential use in the diagnosis and surveillance for swine and other animal diseases.