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Serological evidence of MERS-CoV and HKU8-related CoV co-infection in Kenyan camels - PubMed

Serological evidence of MERS-CoV and HKU8-related CoV co-infection in Kenyan camels

Wei Zhang et al. Emerg Microbes Infect. 2019.

Abstract

Dromedary camels are important reservoir hosts of various coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) that cause human infections. CoV genomes regularly undergo recombination during infection as observed in bat SARS-related CoVs. Here we report for the first time that only a small proportion of MERS-CoV receptor-binding domain positive (RBD) of spike protein positive camel sera in Kenya were also seropositive to MERS-CoV nucleocapsid (NP). In contrast, many of them contain antibodies against bat HKU8-related (HKU8r)-CoVs. Among 584 camel samples that were positive against MERS-CoV RBD, we found only 0.48 (8.22%) samples were also positive for NP. Furthermore, we found bat HKU8r-CoV NP antibody in 73 (12.5%) of the MERS-CoV RBD positive and NP negative samples, yet found only 3 (0.43%) of the HKU8r-CoV S1 antibody in the same samples. These findings may indicate co-infection with MERS-CoV and a HKU8r-CoV in camels. It may also raise the possibility of the circulation of a recombinant coronavirus virus with the spike of MERS-CoV and the NP of a HKU8r-CoV in Kenya. We failed to find molecular evidence of an HKU8r-CoV or a putative recombinant virus. Our findings should alert other investigators to look for molecular evidence of HKU8r-CoV or recombinants.

Keywords: HKU8; MERS; bat; camel; coronavirus.

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Conflict of interest statement

No potential conflict of interest was reported by the authors.

Figures

Figure 1
Figure 1

. Absence of MERS-CoV nucleotide protein (NP) antibody in most of the MERS-CoV receptor-binding domain (RBD) positive camel sera. (A) ELISA screening of MERS-CoV RBD and NP antibodies (n = 891 camels). Cutoff (dashed line, same as below) shown as five times the mean value of the negative samples, it was further adjusted to 0.35 for RBD in line with results from neutralization assay. (B) Luciferase Immunoprecipitation System (LIPS) test for NP antibodies. Eight NP ELISA positive and 12 negative samples (all MERS-CoV RBD positive) were used in the LIPS test. Five RBD and NP ELISA negative samples were used as controls (shown as blue dots), and alpaca anti-serum against MERS-CoV was used as positive controls (red dots). Cutoff was determined as the sum of mean value plus three times the standard deviations of the negative samples. (C) Western blot using whole virus proteins. Lysates of MERS-CoV infected Vero cells were separated on 12% polyacrylamide gel, transferred to nitrocellulose membranes and analysed with MERS-CoV RBD positive, NP positive/negative camel serum samples, or NP mAb. MP, membrane protein. Molecular markers were indicated (kDa). Information for sample applied to WB, LIPS or ELISA can be found in Supplementary Table 1.

Figure 2
Figure 2

. Presence of HKU8r-CoV antibodies in MERS-CoV RBD positive camel sera. (A) Coronavirus NP protein array. Successful expression was determined using anti-HIS tag (all CoVs except HKU2-CoV) or anti-FLAG tag (HKU2-CoV) antibodies. Samples seropositive (C0184) or negative (C0039) for MERS-CoV NP were used. Markers were indicated (kDa). (B) LIPS test for HKU8r-CoV (China strain) NP and S1 antibodies. Twelve NP ELISA positive and eight negative samples (all MERS-CoV RBD positive) were used in LIPS test. Five MERS-CoV RBD and NP ELISA negative samples were used as control in each panel (shown as blue dots), and rabbit anti-serum against HKU8r-CoV S1 protein was used as positive control (red dot). Cutoff was determined as the sum of mean value plus three times the standard deviations of the negative samples. (C) ELISA screening for HKU8r-CoV (China strain) NP and S1 antibody in MERS-CoV RBD positive camel serum samples (n = 584). Cutoff shown as five times the mean value of the negative samples. Information for sample applied to WB, LIPS or ELISA can be found in Supplementary Table 1.

Figure 3
Figure 3

. Venn diagram for virus serum positivity of MERS-CoV or HKU8r-CoV. Totally 605 out of 891 camel sera that were positive for at least one of the viral proteins were included.

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