PHE-CoV belongs to group 2 of the Coronaviridae family, a group characterized by the presence of a gene encoding the HE protein
. Nucleotide sequence analysis of the region covering the S2 probe revealed 92.6% nucleotide sequence homology to BCV and 91.9% homology to HCV-OC43
. Although PHE-CoV causes two distinct clinical syndromes in pigs, only one serotype of the virus is known to exist. Outbreaks of PHE-CoV-associated disease are now on the rise in many countries, inflicting considerable economic damage to the pig industry. Remarkably, these recent isolates showed a high degree of genetic and antigenic homology with the 1962 reference strain PHE-CoV-67 N
. Currently, immunohistochemistry (IHC) for PHE-CoV, or molecular tools such as RT-PCR, enable the detection of specific CoV RNA sequences from infected tissues
[15, 28]. However, these detection techniques usually require special primers, a working laboratory, skilled technicians, and specialized equipment, making rapid and on-site detection of viruses in the field difficult. Therefore, the development of a sensitive, specific, and easily performed assay is crucial for the rapid detection and surveillance of PHE-CoV infection and transmission.
Colloidal gold immunochromatographic assay (GICA) is convenient, is rapid, has high specificity and sensitivity, and can be performed either without instruments or with only a simple instrument, making it suitable for clinical diagnosis and drug testing purposes in almost any context
[29–31]. Colloidal gold is a negatively charged hydrophobic rubber particle that maintains a stable colloidal system through electrostatic repulsion
. The key to successful colloidal gold labeling lies in the preparation of a homogeneous mixture of monodispersed colloidal gold particles
. The microwave oven method was chosen in this experiment because it provides even heating without manual shaking, creating colloidal gold particles that are consistent in size and uniformly distributed, with a mean diameter of about 30 nm under transmission electron microscope. In the preparation of gold markers, proteins and gold glue particles should also be present in an appropriate ratio. If too many antibodies are added, the amount of free antibodies instead of gold-labeled antibodies will increase, which will result in a decreased titer of gold-labeled MAb 4D4, affecting the test’s sensitivity. Conversely, if too few antibodies are added, it is easy for the colloidal particles to aggregate and precipitate, especially during purification by ultracentrifugation. Therefore, it is essential to add sufficient colloidal gold antibodies to maintain stability.
The specificity and sensitivity of the immunochromatographic strip are largely dependent on the following factors. First, the quality of the MAbs used in the strip test is crucial for the specificity and sensitivity of the strip. In this experiment, both of the MAbs used recognize the HE and S proteins of PHE-CoV and have a high affinity for their respective antigen epitopes. Analysis of the specificity showed the strip to be specific for the detection of PHE-CoV, because it reacted with neither two coronaviruses (TGEV and PEDV) in pigs nor with other group 3 viruses of the Coronaviridae family (BCV, HCV-OC43 and MHV), the common virus HCV, or PRV, to which the clinical symptoms of PHE-CoV are similar. In addition, careful selection of a membrane is critical for high specificity, sensitivity, and rapid detection as the wicking rate and speed of liquid diffusion on the membrane (Millipore Corp SHF135, a liner). The membrane used in this experiment was nitrocellulose membrane (Millipore Corp SHF135, a liner) with a membrane pore size of 0.5-1.0 μm, IgG binding force constant of 60–100 μg/cm2, and a chromatography rate of 15–20 s/cm. The membrane ensures antibody absorption and the leaching and binding of gold-labeled proteins. These advantages enable the test results to be sensitive and easy to read
PHE-CoV is able to replicate in the upper respiratory tract with or without producing clinical signs. PHE-CoV can be isolated from the nasal cavity, trachea, brain, and lungs of diseased or healthy pigs
[34–36] and the brain had the highest detection rate by nested PCR and RT-PCR
. Because of RT-PCR and ELISA are also widely applied in clinical practice due to its high sensitivity and specificity
[37, 38]. Thus, a lot of brain tissue samples were collected from deceased piglets with suspected PHE-CoV infection, and using RT-PCR and ELISA as reference test, the relative specificity and sensitivity of the immunochromatographic strip were determined to be 100% and 97.78%, respectively. There was an excellent agreement among the immunochromatographic strip to ELISA and RT-PCR (Kappa = 0.976). The quantifying test result indicated the sensitivity of the strip test to be close to, or slightly less than, that of ELISA and RT-PCR. However, the ELISA and RT-PCR assay each step of the procedure can strongly influence the result if not performed well. The procedure must be strictly followed, with good quality control inside and outside the laboratory, and reagents that ensure the test quality must be selected. Although ELISA and RT-PCR was developed for the detection of PHE-CoV with high specific and sensitivity, it is not suitable for use outside of the research laboratory. In contrast, the procedure for the immunochromatographic strip described in this paper is less laborious and time-consuming than the ELISA and RT-PCR method. Taken together, the results suggested that an immunochromatographic strip was developed with high sensitivity and specificity for detecting PHE-CoV, which could be used for clinical applications.
PHE-CoV is excreted for 8–10 days in oronasal secreions
[34, 39], with transmission occurring through exposure to nasal secretions. Thus, a total of 468 nasal cavity or throat swabs were collected from approximately 1- to 3-week-old piglets, with vomiting and neurological symptoms consistent with PHE-CoV infection, and application of the immunochromatographic strips for diagnosis of PHE-CoV infection in the Jilin province. The positive rate ranged from 61.54% in the Jilin district to 17.95% in the Songyuan district. Notably, the PHE-CoV positive rate was 41.03% in Liaoyuan district in the current study. However, the serum antibody positive rate was only 6.5% in the previous study
. This may be associated with the samples collected from different herds. These findings suggest that PHE-CoV is commonly transmitted in these districts in the Jilin province. Notably, specific pathogen-free (SPF) pigs, derived from germ-free pigs and given artificial milk, have been introduced extensively in many farms in this area
[15, 41]. Because these animals lack antibodies to certain pathogenic agents, including PHE-CoV, their susceptibility to these agents may be greater. Therefore, reliable testing methods are important for the diagnosis and prevention of PHE-CoV disease, which can cause fatal outbreaks in PHE-CoV seronegative farms.