Immunoreactivity characterisation of the three structural regions of the human coronavirus OC43 nucleocapsid protein by Western blot: Implications for the diagnosis of coronavirus infection

Abstract

Previous studies have reported that a prokaryotic-expressed recombinant nucleocapsid protein (NP) is a suitable reagent for the epidemiological screening of coronavirus infection. In this study, soluble recombinant human coronavirus OC43 (HCoV-OC43) NP was produced to examine the antigenicity of the HCoV-OC43 NP of betacoronavirus. Using the purified recombinant NP as an antigen, a polyclonal antibody from rabbit serum with specificity for HCoV-OC43 NP was generated; this antibody reacts specifically with HCoV-OC43 NP and does not cross-react with other human CoV NPs (including those of SARS-CoV and HCoV-229E) by Western blot. Sera from 26 young adults, 17 middle-aged and elderly patients with respiratory infection, and 15 cord blood samples were also tested. Strong reactivity to the NPs of HCoV-OC43 was observed in 96%, 82%, and 93% of the serum samples from the young adults, respiratory patients, and cord blood samples, respectively. To identify the immunoreactivities of the three structural regions of the NP that are recognised by the rabbit polyclonal antibody and human serum, the antigenicities of three protein fragments, including the N-terminal domain (aa 1-173), the central-linker region (aa 174-300), and the C-terminal domain (aa 301-448), were evaluated by Western blot. The rabbit polyclonal antibody demonstrated greater immunoreactivity to the central-linker region and the C-terminal domain than to the N-terminal domain. Three different patterns for the immunoreactivities of the three structural regions of HCoV-OC43 NP were observed in human serum, suggesting variability in the immune responses that occur during HCoV-OC43 infection in humans. The central-linker region of the NP appeared to be the most highly immunoreactive region for all three patterns observed. The goal of this study was to offer insight into the design of diagnostic tools for HCoV infection.

A systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease

“…While exact dynamics from 6 months to 20 years vary between studies, the general trend remains consistent. Figure 7 displays HCoV ageseroprevalence curves for the 7 papers with digitizable data on seroprevalence by age, with the panels representing the four major endemic strains 74,[110][111][112][113][114][115] . Trends from papers not displayed in the figure are largely the same 68,70,116,117 .…”

“…The review gave rise to several observations that were either consistent or conflicting across papers. Studies that enrolled children less than 6 months detected loss of maternal antibodies, representing a preliminary line of defense for newborn children 70,71,74,117 . Most studies showed no discernible difference in age-seroprevalence trends or in overall seroprevalence by strain.…”

Analysis of preferred codon usage in the coronavirus N genes and their implications for genome evolution and vaccine design

“…Studies reported that the N protein produced from prokaryotes has been used to generate specific antibodies against various animal coronaviruses including SARS (Loa et al, 2004;Timani et al, 2004;Wu et al, 2004aWu et al, , 2004bBlanchard et al, 2011). The recombinant antigenic N protein from hCoV OC43 used against the rabbit polyclonal antibodies specific for hCoV OC43 and did not crossreact with other coronaviruses (SARS CoV and hCoV 229E) (Liang et al, 2013). Moreover, it was tested in different aged human serum samples and exhibited strong reactivity due to the effective central portion (174-300 amino acids) of the N protein followed by C (301-448) & N (1-173) terminal portions Yu et al, 2008;Liang et al, 2013).…”

“…The recombinant antigenic N protein from hCoV OC43 used against the rabbit polyclonal antibodies specific for hCoV OC43 and did not crossreact with other coronaviruses (SARS CoV and hCoV 229E) (Liang et al, 2013). Moreover, it was tested in different aged human serum samples and exhibited strong reactivity due to the effective central portion (174-300 amino acids) of the N protein followed by C (301-448) & N (1-173) terminal portions Yu et al, 2008;Liang et al, 2013). Hence the N protein functions as a sensitive and specific diagnostic tool for hCoV OC43 (Di et al, 2005;He et al, 2005) and it has been further useful in the detection of SARS CoV infection (after the first day of infection) (Che et al, 2004).…”

Crystal structure-based exploration of the important role of Arg106 in the RNA-binding domain of human coronavirus OC43 nucleocapsid protein

“…It has also been shown to act as an RNA chaperone [21]. Moreover, the N protein is an important diagnostic marker and is an antigen for the host antibody and T cell immune responses [16,[22][23][24].…”