Study On Antigenic Relationship Between Human Coronavirus N Protein And Antibody Serum Prevalence In Healthy Population

As major causative agents of acute respiratory tract diseases, human coronavirus (HCoV) attracts more and more attention since the outbreak of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) in recent years. Six HCoV-NL63,229E, OC43, HKU1, SARS-CoV, and MERS-CoV have been identified thus far. NL63and229E belong to alphacoronavirus (a), and OC43, HKU1, SARS-CoV, and MERS-CoV belong to betacoronavirus (β). However, the antigenic and epidemiologic properties of HCoV in the population are not fully understood. In this study, we established a competitive ELISA method to analyze the antigenic and serological relationships among nucleocapsid proteins (N proteins), which were expressed at the highest level and the most commonly used in serological diagnosis, and the epidemiology of the six HCoV in the population were analyzed by detecting the IgG antibodies against N proteins.Firstly, prokaryotic expression system was used to express N proteins of HCoV. Recombinant N proteins were purified with a purity>90%by cation exchange and Ni2-metal chelate chromatography. Then the normal human serum samples were used for screening positive and negative reference serum samples. The cross-reactivities among HCoV N proteins were analyzed by competitive ELISA. The results showed that the cross-reactivities existed within N proteins of subgenuses (NL63and229E, OC43and HKU1), but no cross-reactivity across subgenuses.In order to avoid interference caused by cross-reactivities, we established a competitive ELISA method using N proteins of HCoV. On this basis, IgG specific antibodies against six HCoV N proteins were deteted in serum samples from healthy individuals. The results showed that SARS-CoV and MERS-CoV antibodies were not detected, while antibodies against N proteins of NL63,229E, OC43and HKU1were found widely existing in the population. Low seroprevalence rates,12.9%,0,12.9%and3.2%for NL63,229E, OC43and HKU1, respectively, were detected in the infants under6months. While the seroprevalence rates increased with ages and reached to39%,18.1%,46.2%and17%in children of2-5years old group. Then the seroprevalence rates up to peak in15-44age group, respectively, with67.1%,50.85%,70.8%and25.6%for NL63,229E, OC43and HKU1, respectively. After that they decreased to25.2%,10.7%,40.3%and14.5%, respectively, in≥60years age group. In addition, the seroprevalence rates of NL63and OC43were obviously higher than that of229E and HKU1, this result suggested that there were differents in infectious and epidemic levels in four commoun HCoV, there were no antibodies of SARS-CoV and MERS-CoV in ous serum samples.In order to evaluate the correlation between serum IgG antibody levels and acute phase of infection, IgG antibodies against six HCoV N proteins were detected with the serum samples which corresponding respiratory samples were diagnosed positive and negative by PCR assay. The results showed that the positive rates of PCR negative serum samples were higher than that of PCR positive samples, this result indicated that N protein antibodies might negatively correlate with infection of HCoV.In summary, our study suggests that the cross-reactivities within groups should be taken into consideration when testing the specific antibodies against N proteins. However, the common antibody detection should include N antigens across different groups. Our data suggested a differential prevalence of HCoV in healthy individuals. The seroprevalences for NL63and OC43among healthy population were significantly higher than that of229E and HKU1. However, none of the healthy population was shown to have IgG antibody to SARS-CoV and MERS-CoV, our country should strengthen the prevention and control measures. The IgG antibody levels of N protein was negatively correlated with HCoV infection. This study provided a foundation for understanding the characteristics of immunological response of HCoV infection, developmenting diagnostic reagents and evaluating epidemic spread of HCoV in our country.