Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic disease that was first identified in humans in 2012 in Saudi Arabia. MERS-CoV causes acute and severe respiratory disease in humans. The mortality rate of MERS in humans is ∼35% and >800 deaths have been reported globally as of August 2020. Dromedary camels are a natural host of the virus and the source of zoonotic human infection. In experimental studies, Bactrian camels are susceptible to MERS-CoV infection similar to dromedary camels; however, neither the virus, viral RNA, nor virus-specific antibodies were detected in Bactrian camel field samples so far. The aim of our study was to survey Mongolian camels for MERS-CoV–specific antibodies. A total of 180 camel sera, collected in 2016 and 2017, were involved in this survey: 17 of 180 sera were seropositive with an initial enzyme-linked immunosorbent assay (ELISA) test performed at the State Central Veterinary Laboratory in Mongolia. These 17 positive sera plus 53 additional negative sera were sent to the Rocky Mountain Laboratories, NIAID/NIH, and tested for the presence of antibodies with a similar ELISA, an indirect immunofluorescence assay (IFA), and a virus neutralization test (VNT). In these additional tests, a total of 21 of 70 sera were positive with ELISA and 10 sera were positive with IFA; however, none was positive in the VNT. Based on these results, we hypothesize that the ELISA/IFA-positive antibodies are (1) non-neutralizing antibodies or (2) directed against a MERS-CoV-like virus circulating in Bactrian camels in Mongolia.
Introduction
Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic agent that has caused >800 fatalities and ∼2500 human cases in 27 countries so far (World Health Organization); the virus was first identified in a human patient in 2012 (Zaki et al. 2012). Human-to-human transmission of MERS-CoV has been previously described, and the main source of human infections are dromedary camels (C. dromedarius), which are a natural reservoir of the virus (Younan et al. 2016). Surveillance of dromedary camels for the presence of MERS-CoV–specific antibodies indicated a high percentage of seropositivity among dromedary camels in the Middle East and in north and east Africa (Harcourt et al. 2018, Hemida et al. 2014). Likewise, widespread presence of MERS-CoV or MERS-CoV RNA was detected in dromedary camels in the Middle East and in Africa (van Doremalen et al. 2017, Sikkema et al. 2019).
Bactrian camels consist of two subspecies: wild Bactrian camels (Camelus bactrianus ferus) and domesticated Bactrian camels (Camelus bactrianus bactrianus); they are found throughout central, western, and southern Asia. Interestingly, the distribution of Bactrian camels overlaps geographically with dromedary camels (Kaoru et al. 2017). However, neither MERS-CoV genomic RNA nor specific antibodies have been detected in Bactrian camels yet, despite a recent study demonstrating susceptibility of Bactrian camels to experimental MERS-CoV infection (Adney et al. 2019). The presence of MERS-CoV–specific antibodies and viral RNA was investigated in 2014 using 200 Bactrian camels located in three soums (i.e., districts) within the Umnugovi and Dundgovi provinces (Fig. 1) in Mongolia, but no evidence of MERS-CoV circulation was found (Chan et al. 2015). In addition, serosurveillance for MERS-CoV in both Bactrian and dromedary camels in Kazakhstan did not detect any evidence for circulating MERS-CoV (Miguel et al. 2016).
Camel density and sampling sites (number of camels per 100 km2).
Materials and Methods
Study population and sampling
In this article, we describe a serosurveillance study that was conducted in Mongolia from September 2016 to September 2017. Blood was collected from domesticated Bactrian camels in the Khuld and Noyon soums within the Dundgovi and Umnugovi provinces (Fig. 1), respectively. Sera were obtained and stored in the serum bank of the State Central Veterinary Laboratory (SCVL), Mongolia. The Umnugovi and Dundgovi provinces have the highest and second highest density of Bactrian camels in Mongolia, respectively (Fig. 1). These studies were approved by the Mongolian Ministry of Education and Science.
Three antibody detection systems were applied to detect MERS-CoV–specific antibodies: indirect enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and virus neutralization test (VNT). The sensitivity and specificity of the MERS ELISA are described in detail by Trivedi et al. (2018). The IFA is a commercial test available from Euroimmun (
Enzyme-linked immunosorbent assay
Maxisorp (Nunc) plates were coated with the MERS-CoV S1 protein (Sino Biological) and incubated at 4°C overnight. Afterward, the plates were blocked with 5% milk in PBS-T and diluted camel sera (1:100) were added into the plate wells. MERS-CoV S1–specific antibodies were detected using anti-llama IgG (H+L) HRP-conjugated antibody (Alpha Diagnostic). Optical density (OD) was measured at 450 nm after 3,3′,5,5′-tetramethylbenzidine substrate and stop solution (Seracare) were added to the plate. Different incubation times were used for the development of the ELISA reaction: 5 min at SCVL and an OD of 1.0 for the positive serum control at the RML. Also, the cutoff for positive samples in the ELISA at the SCVL was the average OD plus 3 × SD and at the RML the cutoff was average OD × 1.5 of negative sera obtained from camels raised in captivity in the United States (van Doremalen et al. 2017). This assay was used in the laboratories of SCVL and the RML, respectively. Positive and negative control sera from MERS-CoV-infected and uninfected camels were utilized as respective controls in all diagnostic tests.
Immunofluorescence assay
IFAs (Euroimmun, FI 2604–1005 G) were performed with camel sera using manufacturer's instructions, but the secondary antibody was replaced with anti-llama IgG (H+L) HRP-conjugated antibody (Alpha Diagnostic). Serum samples tested on IFA were either positive in the ELISA or had OD values just below the cutoff for the ELISA.
Virus neutralization assay
Sera were heat inactivated (30 min, 56°C) and twofold serial dilutions were prepared in 2% DMEM. Hereafter, 100 TCID50 of MERS-CoV were added. After 60 min incubation at 37°C, the virus–serum mixture was added to VeroE6 cells and incubated at 37°C and 5% CO2. At 5 dpi, cytopathic effect was scored. The virus neutralization titer (NT100) was expressed as the reciprocal value of the highest dilution of the serum, which still inhibited 100% of virus replication.
Results
As a result of initial serology testing in Mongolia, 17 of 180 Bactrian camel sera were positive with the MERS-CoV S1 antigen (Table 1). Seventy sera from that collection were sent to the RML (NIAID/NIH), including the 17 ELISA-positive sera and 53 randomly selected ELISA-negative sera.
Serosurveillance of Bactrian Camels for Middle East Respiratory Syndrome Coronavirus
Initial testing of samples was done in Mongolia (SCVL) using the MERS-CoV–specific S1 protein ELISA. Additional testing of samples was performed at the RML using the MERS-CoV–specific S1 antigen ELISA, indirect IFA, and VN tests.
ELISA, enzyme-linked immunosorbent assay; IFA, immunofluorescence assay; MERS-CoV, Middle East respiratory syndrome coronavirus; RML, Rocky Mountain Laboratories; SCVL, State Central Veterinary Laboratory; VN, virus neutralization.
Of the 17 positive samples, 16 were confirmed to be positive by the RML ELISA. Of these samples, eight were also found to be positive by IFA; however, none was positive for neutralizing antibodies. Interestingly, the ELISA performed at RML identified a further five positive serum samples, all of which were negative on IFA and VN assays, respectively. Two serum samples that were negative in both ELISA tests were positive in the IFA. The two ELISA-negative samples that were positive in the IFA had borderline OD values. The 26 serum samples tested on IFA were either positive in the ELISA or had OD values just below the cutoff value.
Discussion
The results reported here with Bactrian camel sera obtained independently in two different laboratories clearly indicate that Mongolian Bactrian camels carry antibodies that specifically recognize MERS-CoV–specific antigens; this was determined using two independent assay formats: one using the recombinant MERS-CoV S1 protein (ELISA) and the other with native MERS-CoV proteins (IFA). The ELISA results performed with the same ELISA system in the two laboratories were not in 100% agreement. This was most likely due to the fact that (1) the cutoff for positive samples in the ELISA at SCVL was the average OD plus 3 × SD, and at the RML, the cutoff was the average OD × 1.5 and (2) different incubation times were used for the development of the ELISA reaction, namely 5 min at SCVL and an OD of 1.0 for the positive serum control at the RML. These data suggest that the MERS-CoV–specific antibodies in Bactrian camels were induced by an MERS-like CoV. Since the ELISA- and IFA-positive sera were negative by the VN test using a currently circulating MERS-CoV strain, we hypothesize that the Bactrian camel sera either do not have neutralizing antibodies against the MERS-CoV strain used in the VN tests or the virus that infects Bactrian camels is a MERS-like CoV or another camel betacoronavirus that is not identical to the MERS-CoV circulating in the Middle East. In a previous study, Chan et al., in Mongolian camels, did not detect MERS-CoV-specific antibodies. This could be due to (1) the respective antibody test systems employed here are more sensitive than the MERS-CoV pseudovirus neutralization tests employed by Chan et al.; (2) the timing of sampling (2014 vs. 2016/2017); or (3) the camel herds that were sampled.
Further serological and molecular surveillance, specifically in young camels, and an extended number of samples from other provinces with high density of camel populations in Mongolia, for example, Uvurkhangai, Bayankhongor, and Dornogovi provinces, will be necessary to determine whether MERS-CoV or a similar virus circulates within the Bactrian camel population in Mongolia.
Footnotes
Author Disclosure Statement
No competing financial interests exist
Funding Information
This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), and by NBAF Transition Funds from the state of Kansas.