A total of 22 samples were found positive by two independent RT-qPCR assays (e.g. N2 and N3 RT-qPCR), though with high Ct values > 35. Additionally, from one of these samples, we were able to amplify partial Nucleocapsid and Spike gene segments (GenBank accession nos: MH102354 and MH102355), with a size of 228 bp and 960 bp, respectively. The sequences obtained from the Pakistani camel were identical with several already published sequences obtained from camel as well as humans from the Arabian Peninsula. Unfortunately, likely due to RNA degradation we were unable to recover additional sequences but are confident in the fidelity of the finding. First, viral RNA extraction and RT-qPCR experiments were performed in a laboratory where no previous MERS-CoV work has ever been done. Second, hemi-nested PCR for N gene were repeated in two independent laboratories by two different persons yielding identical results. These results indicate active circulation of closely-related or identical strains circulating in Pakistan compared to the Arabian Peninsula.
For serology, 1, 050 camel serum samples were collected, out of these 695 (66.19%) were females and 355 (33.81%) were from male camels. The majority of sera were from Punjab Province (57.14%) and semi-nomads (37.10%). The distribution of sera by age, sex, type of herd and sampling location is presented in Table 1. Of 1, 050 camel sera tested by ELISA 794 (75.62%) sera were found to be positive by ELISA (Figs. 1, 3). Slightly higher prevalence was observed in camels from Khyber Pakhtunkhwa (KPK) (79.76%, 95% CI 72.72–85.40) compared to Balochistan (77.13%, 95% CI 70.33–82.80), Punjab (74.50%, 95% CI 70.77–77.90) and Sindh (72.34%, 95% CI 61.95–80.83); however, the differences were not significant. Prevalence increased with the age and the highest seroprevalence was recorded in camel aged > 10 years (81.37%, 95% CI 74.31–86.89) followed by those aged 3.1–10 years (78.65%, 95% CI 75.42–81.57) and ≤ 3 years (58.19%, 95% CI 50.54–65.48). The age of camel was the main determinant of prevalence as older animals (> 10 years) were three times more likely to be positive (Odds Ratio 3.13, 95% CI 1.86–5.35) as compared to younger animals (≤ 3 years). Significantly (P < 0.001) higher prevalence was observed in females (78.13%, 95% CI 74.83–81.11) as compared to males (70.70%, 95% CI 65.62–75.33). Significantly, higher prevalence was observed in nomadic camels (89.17, 95% CI 82.97–93.38) followed by pastoralists (78.19%, 95% CI 73.19–82.51), semi-nomads (71.46%, 95% CI 66.65–75.85) and sedentary (68.31%, 95% CI 60.96–74.86) (Table 1).
Table 1. Univariate analyses of Middle East Respiratory Syndrome Coronavirus ELISA-positive camels with their determinants.
Figure 3. Histogram displaying the frequency distribution of MERS-CoV IgG ELISA optical density (OD) ratios. A ELISA OD ratios for dromedary camels tested in this study. B ELISA OD ratios for dromedaries used to determine the sensitivity and specificity. The vertical dashed lines represent the ELISA cut-off values.
To further verify the ELISA results, 100 ELISA positive and 20 ELISA negative samples were randomly chosen and tested for MERS-CoV neutralizing antibodies by microneutralization assay as described elsewhere (Perera et al. 2013). Neutralization assay results were found to be in agreement with the ELISA results, further confirming the sensitivity and specificity of in-house ELISA.
In total, 2, 409 human sera samples were collected from four provinces of Pakistan during 2016–2017. Out of 2, 409 samples, 1, 249 were from females and 1, 160 were from males. Most of the sampled population was involved in livestock rearing. Among the sampled population, 840 humans were camel herders.
A total of 91 human sera samples with OD values ≥ 0.2, which is as high as three times the ratio of the mean value of all tested human serum samples without known exposure to camels, were further tested with a commercially available ELISA kit (Euroimmun, Lubeck, Germany). Thirtysix samples were found to be positive by commercial ELISA, all having OD values ≥ 0.35 (Fig. 4). All the reactive samples were from Punjab Province of Pakistan. Of note, out of these 36 ELISA positives, 30 of them were from camel herders. To further confirm the presence of antibodies in these samples, all 91 sera were tested for MERS-CoV neutralizing antibodies by microneutralization assay as described elsewhere (Perera et al. 2013). None of them was positive.