Virus isolation (VI) is the most reliable method for detecting BVDV and is considered the “gold standard” for BVDV diagnosis (Sandvik, 2005). Primary cells such as bovine uterine endometrial cells (Cheng et al., 2017), bovine testis (BT) cells (Weber et al., 2017), bovine kidney (BK) cells (Suda et al., 2019), bovine turbinate (BTu) cells (Falkenberg et al., 2021), bovine bronchial epithelial (BBE) cells (Su et al., 2021), bovine lung (BL) cells (La Polla et al., 2022a,b), and bovine skin fibroblast (BSF) cells (Workman et al., 2023) can be used for the isolation of BVDV. Among these, BK, BT, and BTu cells are currently the most widely used primary cells for BVDV isolation. In addition, BVDV isolates can be cultured in vitro in Madin-Darby bovine kidney (MDBK), bovine tonsil (BoTur), and baby hamster kidney (BHK) cell lines. MDBK is more susceptible to BVDV infection (Odeón et al., 2009), and is commonly used for BVDV culture (Workman et al., 2021; Yang et al., 2021; Wang et al., 2023).

The VI assay is highly accurate and does not require complex instruments, but some problems should not be underestimated. First, virus isolation can only identify cp BVDV, but not ncp BVDV. Second, this method is labor intensive, time-consuming, and not suitable for processing a large number of samples. Third, the final judgment of BVDV infection tends to be influenced by several factors, such as the sensitivity of inoculated cells and the way the sample is preserved or transported.

Enzyme-linked immunosorbent assay (ELISA) is a rapid and high-throughput serological diagnostic method for the detection of a specific antigen or antibody from certain pathogens. The main types of ELISA include direct, indirect, double-antibody sandwich, and competitive methods (Tabatabaei and Ahmed, 2022). Zhang et al. used a purified recombinant BVDV-E2 protein to immunize chickens and acquired the specific E2-IgY antibody from egg yolk. A total of 22 blood samples from diarrheic cattle were collected to assess the indirect ELISA assay based on E2 IgY and the assay yielded 95.45% concordance with the RT-PCR assay for detecting BVDV (Zhang et al., 2016). Furthermore, Marzocca et al. developed an indirect ELISA for the detection of BVDV using a recombinant C-terminal truncated E2 protein (tE2) expressed in the Drosophila melanogaster system. The tE2 protein was efficiently secreted in the supernatant in the form of post-translational modification, without the need for purification. The initial examination of 183 cattle serum samples utilizing the tE2-ELISA demonstrated a specificity of 98% and a sensitivity of 100% compared to the standard BVDV neutralization test (Marzocca et al., 2007). Shapouri et al. developed a competitive ELISA (cELISA) for detecting BVDV antibodies in serum samples. The NS3 protein, which was highly conserved among pestiviruses, was used as a recombinant antigen in combination with a monoclonal antibody as a competitive antibody. The cELISA assay was used to analyze a total of 197 serum samples, demonstrating a sensitivity of 93.90% and a specificity of 100% when compared to the virus neutralization test (Shapouri et al., 2022). Moreover, a multiplex indirect ELISA assay was developed to identify antibodies against five viral pathogens, including bovine respiratory syncytial virus (BRSV), bovine herpes virus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), parainfluenza virus type 3 (PI3V), and enzootic bovine leukosis virus (EBLV) using complete viruses of BRSV and BoHV-1, and recombinant E2/BVDV, HN/PI3V, and gp51/EBLV as capture antigens. Diagnostic agreement for samples that were analyzed concurrently using monoplex and multiplex assays was nearly perfect for BoHV-1, EBLV, BRSV, and BVDV (above 0.81) and substantial for PI3V (ranging from 0.61 to 0.80) (Rodriguez et al., 2023). In summary, ELISA is the most commonly used technique in serology due to its high specificity, high sensitivity, good reproducibility, low cost, and ease of operation, making it a ideal choice for high-throughput applications.

Immunofluorescence assay (IFA) is a technique in which a fluorescent antibody or antigen is used as a probe to detect an unknown antigen or antibody (Piña et al., 2022). Bedeković et al. developed an IFA method for the diagnosis of BVDV in which a specific monoclonal antibody for BVDV was used as primary antibody and an anti-mouse antibody conjugated with fluorescein isothiocyanate (FITC) as secondary antibody. The IFA assay showed a sensitivity and specificity of 100% in detecting 9 positive samples when compared to the RT-PCR assay (Bedeković et al., 2011). Yang et al. generated BVDV E0 + E2 or E2 + E2 virus-like particles (VLPs) using an insect baculovirus expression vector system. The expression of E0 + E2 and E2 + E2 were subsequently detected by immunofluorescence assay (IFA) and western blot (Yang N. et al., 2022). In another experiment, the IFA was applied to detect a positive BVDV-3 strain using BVDV-positive serum as primary antibody and FITC-labeled rabbit anti-bovine IgG as secondary antibody. MDBK cells infected with the BVDV-3 strain were observed to exhibit green fluorescence while uninfected cells did not (Yang et al., 2023). IFA has the advantages of good sensitivity and specificity, but non-specific staining phenomenon occurs, and it needs to be observed with the help of a special fluorescence microscope, which is costly and unsuitable for the promotion of the application at the grass-roots level.

Immunohistochemistry (IHC) is a technique of detecting antigen in tissues by a chemical reaction that results in the coloration of a chromogenic agent based on the binding of antibodies to a specific antigen in tissue sections (Harms et al., 2023). In a previous study, a total of 184 cattle underwent immunohistochemical tests on skin biopsies to determine the presence of BVDV infection. The IHC assay was determined to be sensitive and specific in detecting BVDV infection (Thür et al., 1996). Bianchi et al. used oral mucosa, skin tissue, and small intestine sections from three calves that died from BVDV infection for the IHC assay. The results showed notable immunostaining within the cytoplasm of epidermal and follicular epithelial cells, indicating the presence of the BVDV antigen within the cytoplasm of skin and mucosal epithelial cells (Bianchi et al., 2017). The IHC assay is sensitive, rapid, and can be applied to a large number of samples. Furthermore, the method is not interfered by maternal antibodies in blood samples and is therefore widely used in the detection of PI animals (Brodersen, 2004; Hilbe et al., 2007). However, IHC is a labor intensive test that is prone to operational problems and has more subjective influences on the criteria for judging the results, and it requires higher technical experience from personnel (Cornish et al., 2005).

Reverse transcription-polymerase chain reaction (RT-PCR) involves extracting RNA from tissue cells, reverse transcribing it into complementary DNA (cDNA) using reverse transcriptase, and finally using cDNA as a template for PCR amplification (Donovan et al., 2022). The RT-PCR method for detecting BVDV was first established by Hertig et al. in 1991, based on the p80 and gp53 genes that encode the NS3 and E2 protein, respectively (Hertig et al., 1991). Since then, this method has been widely used for the diagnosis of BVDV. Letellier et al. developed an RT-PCR assay based on the highly conserved 5’-UTR region to detect both the BVDV-1 and BVDV-2 genotypes and differentiate them from other plague viruses (Letellier et al., 1999). Monteiro et al. designed and evaluated various primer sets to detect 135 serum samples positive for BVDV antigens by an antigen ELISA using RT-PCR assays. Compared to other primer sets, the newly designed BP189-389 primers had the ability to detect all 135 ELISA-positive samples including BVDV-1 (n = 64), BVDV-2 (n = 45), and HoBi-like pestivirus (n = 26) (Monteiro et al., 2019). Furthermore, several multiplex RT-PCR approaches have been established and utilized for BVDV detection, in addition to single-gene RT-PCR. Lung et al. have developed a highly accurate multiplex RT-PCR and an innovative automated microarray that can simultaneously detect eight viruses that affect cattle, including BVDV-1 and BVDV-2, vesicular stomatitis virus (VSV), bluetongue virus (BTV), malignant catarrhal fever virus (MCFV), bovine herpesvirus-1 (BoHV-1), parapoxvirus (PPV), and rinderpest virus (RPV). The approach accurately identified a panel of 37 strains of the eight target viruses and detected a mixed infection. Limit of detection of the multiplex RT-PCR and microarray assay for BVDV were both 30 TCID₅₀/mL (Lung et al., 2017). RT-PCR is a superior method of detection, offering faster detection, greater specificity, and higher sensitivity compared to virus isolation and IFA. In addition, RT-PCR can determine the nucleic acid sequence of BVDV in samples in which the virus has been inactivated.

Real-time PCR is an appealing alternative to conventional PCR since it can monitor the production of amplification products in real-time through the detection of a fluorescent signal during each cycle of the PCR reaction. Real-time PCR assays are highly specific, sensitive, and highly automated, making them ideal for high-throughput measurements (Bustin, 2005). The SYBR Green I dye and the TaqMan probe are the two most commonly used real-time PCR assays. TaqMan-based real-time PCR is significantly more specific for nucleic acid detection and quantification compared to the SYBR Green I assay, although the cost of synthesizing specific probes is too high for grassroots use.

Digital droplet PCR (ddPCR) is a powerful technique that combines PCR with droplet microfluidics, allowing for the precise quantification of target molecules in a sample. By dropletising the sample before amplification, ddPCR enables the absolute quantification of nucleic acid molecules with unparalleled accuracy and sensitivity (Chen et al., 2021). Flatschart et al. successfully detected BVDV genome copies in cell cultures using ddPCR, demonstrating its potential as a reliable and robust method for nucleic acid analysis. This assay enables the absolute quantification of the BVDV genome without the use of calibration standards (Flatschart et al., 2015). In contrast, ddPCR confidently achieved a limit of detection of 13 copies/μL and a limit of quantification of 38 copies/μL for BVDV, demonstrating its superiority in absolute quantitative studies of BVDV RNA (Henrique et al., 2023). The ddPCR offers significant technical advantages and has promising applications, making it a reliable choice for researchers. First, it allows for absolute quantification of target molecules without the need for standards. Second, it is fast, accurate and effectively avoids false positives and false negatives. Third, the technique is not dependent on amplification efficiency and is particularly suitable for detecting samples with complex matrices. Finally, the results are easy to analyze.

Loop-mediated isothermal amplification (LAMP) is a rapid thermostatic nucleic acid amplification technique developed by Notomi in 2000 for the detection of various infectious diseases (Notomi et al., 2000; Pang and Long, 2023). This multistep reaction is carried out at around 60–70°C using strand-displacing DNA polymerase and 4–6 primers that recognize 6–8 unique sites on the target nucleic acid.

Aebischer et al. developed three highly sensitive assays, including high-speed RT-qPCR, LAMP, and RPA and compared their performance for detecting Schmallenberg virus and BVDV (Aebischer et al., 2014). Furthermore, Fan et al. optimized a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for detecting BVDV-1 and BVDV-2 using six primer pairs that were designed based on the 5’-UTR of the BVDV genome. The method was able to detect 4.67 RNA copies without the need for complex equipment. In addition, it has high specificity and no cross-reactivity with BRV, CSFV, BoHV-1, and BCoV. This method holds great promise for clinical diagnosis and field monitoring of BVDV (Fan et al., 2012). LAMP amplification products are commonly identified through agarose gel electrophoresis. The incorporation of fluorescent dyes into the LAMP product allows for direct visual detection of positive products. In the study conducted by Tajbakhsh et al., GelRed fluorescent dye was used to detect LAMP-amplified BVDV products. Positive samples were observed to have a fluorescent yellow reaction color, while negative samples retained their red reaction color under UV light. This method is advantageous in terms of simplicity, time-saving, and cost-effectiveness, making it suitable for detecting BVDV in the field (Tajbakhsh et al., 2017). However, it should be noted that LAMP is not suitable for detecting multiple pathogens as it cannot accurately identify the effect of a single pathogen among multiple pathogens due to its single expression (Ayaz Kök et al., 2023).

Recombinase polymerase amplification (RPA) is an isothermal amplification system that is highly effective for virus detection, especially for point-of-care testing (POCT) (Munawar, 2022). The RPA reaction uses recombinant enzymes and oligonucleotide primers to form protein-DNA complexes that efficiently search for homologous sequences in double-stranded DNA. By initiating a strand displacement reaction, it rapidly initiates DNA synthesis that exponentially amplifies the target on the template. The single-stranded DNA binding protein (SSB) binds to the replaced DNA strand and forms a D-loop that prevents further substitutions (Li and Macdonald, 2015). The whole amplification process is rapid and reaches detectable levels within minutes. Yang et al. developed a method for the rapid and easy detection of BVDV and BPIV-3 by targeting the BVDV 5’-UTR and the phosphorylated protein P gene of BPIV-3, through the combined application of reverse transcriptase recombinase polymerase amplification (RT-RPA) and lateral flow test paper (LFD). The method amplifies BVDV and BPIV3 RNA by applying RT-RPA within 25 min at 35°C. The reaction products are observed on the LFD within just 5 min at room temperature. The lowest detection limit of 50 copies/μL for BVDV and 34 copies/μL for BPIV-3 further showcase the impressive capabilities of this method. Notably, the method does not cross-react with viruses such as CSFV and IBRV (Yang S. et al., 2022). The RPA technology exhibits a notable level of specificity and sensitivity, undergoes rapid amplification, offers ease of operation, and requires a lower temperature than RT-PCR and LAMP, making it a highly efficient option for virus detection. Nevertheless, the RPA method has limitations, such as the potential for false positives during the detection process (Munawar, 2022).

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) systems have been widely used for genome editing due to its precise recognition and cleavage of specific DNA and RNA sequences. Furthermore, it is important to note that certain CRISPR/Cas systems, including Cas13, Cas12a, and Cas14, possess collateral nonspecific catalytic activities that can be harnessed for nucleic acid detection. This is accomplished by breaking down a labeled nucleic acid to generate a fluorescent signal (Aman et al., 2020).

Hwang et al. developed a highly efficient CRISPR-Cas13 system for point-of-care testing (POCT) of BVDV by precisely targeting the BVDV-1b 5’-UTR. Four specially designed and synthesized CRISPR RNAs (crRNAs) were utilized to ensure accurate targeting of the BVDV-1b 5’-UTR. The expression and purification of the LwCas13a protein was optimized and the RNase activity of LwCas13a was verified. The collateral cleavage activity of Cas13 was then confirmed using a reliable colorimetric lateral-flow detection assay (LFDA) (Hwang et al., 2023). Yao et al. also established a CRISPR-Cas13a system that successfully detected BVDV nucleic acid with a detection limit of 10³ picomolar (pM) using a fluorescence (FAM)- and quencher (BHQ-1)-labeled RNA probe (Yao et al., 2021). The CRISPR-Cas13a system-based nucleic acid detection method for BVDV offers several advantages. These include rapid detection, high sensitivity, good specificity, high-throughput, result visualization, and adaptability to point-of-care testing.