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Background. Annually, Saudi Arabia is the host of the Hajj mass gathering. We aimed to determine the Middle East respiratory syndrome coronavirus (MERS-CoV) nasal carriage rate among pilgrims performing the 2013 Hajj and to describe the compliance with the Saudi Ministry of Health vaccine recommendations. Method. Nasopharyngeal samples were collected from 5235 adult pilgrims from 22 countries and screened for MERS-CoV using reverse transcriptase-polymerase chain reaction. Information regarding the participants' age, gender, country of origin, medical conditions, and vaccination history were obtained. Results. The mean age of the screened population was 51.8 years (range, 18-93 years) with a male/female ratio of 1.17:1. MERS-CoV was not detected in any of the samples tested (3210 pre-Hajj and 2025 post-Hajj screening). According to the vaccination documents, all participants had received meningococcal vaccination and the majority of those from at-risk countries were vaccinated against yellow fever and polio. Only 22% of the pilgrims (17.5% of those ≥65 years and 36.3% of diabetics) had flu vaccination, and 4.4% had pneumococcal vaccination. Conclusion. There was no evidence of MERS-CoV nasal carriage among Hajj pilgrims. While rates of compulsory vaccinations uptake were high, uptake of pneumococcal and flu seasonal vaccinations were low, including among the high-risk population.
Ministry of Health, Jeddah, 4 Saudi Aramco Medical Services Organization, Dhahran, Kingdom of Saudi Arabia; 5 Indiana University School of Medicine, Indianapolis; 6 Special Infectious Diseases Unit

Ebola virus (EboV) belongs to the Filoviridae family of viruses that causes severe and fatal hemhorragic fever. Infection by EboV involves fusion between the virus and host cell membranes mediated by the envelope glycoprotein GP2 of the virus. Similar to the envelope glycoproteins of other viruses, the central feature of the GP2 ectodomain postfusion structure is a six-helix bundle formed by the protein's N-and C-heptad repeat regions (NHR and CHR, respectively). Folding of this six-helix bundle provides the energetic driving force for membrane fusion; in other viruses, designed agents that disrupt formation of the six-helix bundle act as potent fusion inhibitors. To interrogate determinants of EboV GP2-mediated membrane fusion, we designed model proteins that consist of the NHR and CHR segments linked by short protein linkers. Circular dichroism and gel filtration studies indicate that these proteins adopt stable ahelical folds consistent with design. Thermal denaturation indicated that the GP2 six-helix bundle is highly stable at pH 5.3 (melting temperature, T m , of 86.8 6 2.0°C and van't Hoff enthalpy, DH vH , of 228.2 6 1.0 kcal/mol) and comparable in stability to other viral membrane fusion six-helix bundles. We found that the stability of our designed a-helical bundle proteins was dependent on buffering conditions with increasing stability at lower pH. Small pH differences (5.3-6.1) had dramatic effects (DT m 5 37°C) suggesting a mechanism for conformational control that is dependent on environmental pH. These results suggest a role for low pH in stabilizing six-helix bundle formation during the process of GP2-mediated viral membrane fusion.
Designed protein mimics of the Ebola virus glycoprotein GP2 a-helical bundle: Stability and pH effects

A multiplex quantitative real-time polymerase chain reaction (mqPCR) assay was developed and validated for the detection and differentiation of porcine circovirus type 3 (PCV3) and type 2 (PCV2) strains. The assay coverage was 97.9% (184/188) for PCV3 and 99.1% (1889/1907) for PCV2 sequences that were available from the current GenBank database. The PCR amplification efficiencies were 98-99% for plasmids, and 92-96% for diagnostic samples, with correlation coefficients all greater than 0.99. The limit of detection (LOD) determined as plasmid copies per reaction was 17 for PCV3 and 14 for PCV2. The assay specifically detected the targeted viruses without cross reacting to each other or to other common porcine viruses. Among 336 swine clinical samples collected in 2018, 101 (30.1%) were PCV3 positive, 56 (16.7%) were PCV2 positive and 18 (5.4%) were co-positives. Sixty selected PCV3 positives were confirmed by Sanger sequencing, and 53 of the 56 PCV2 positive samples were tested positive by another validated PCR assay.

A multiplex real-time PCR assay for the detection and differentiation of the newly emerged porcine circovirus type 3 and continuously evolving type 2 strains in the United States