Beta interferons from the extant camelids: Unique among eutherian mammals.

The COVID-19 pandemic is a wake-up call on the zoonotic viral spillover events and the need to be prepared for future outbreaks. Zoonotic RNA viruses like the Middle East respiratory syndrome coronavirus (MERS-CoV) are potential pathogens that could trigger the next pandemic. Dromedary camels are the only known animal source of MERS-CoV zoonotic infections, but little is known about the molecular antiviral response in this species. IFN-ß and other type-I interferons provide the first line of defense against invading pathogens in the host immune response. We identified the IFNB gene of the dromedary camel and all extant members of the family Camelidae. Camelid IFN-ß is unique with an even number of cysteines in the mature protein compared to other eutherian mammals with an odd number of cysteines. The viral mimetic poly(I:C) strongly induced IFN-ß expression in camel kidney cells. Induction of IFN-ß expression upon infection with camelpox virus was late and subdued when compared to poly(I:C) treatment. Prokaryotically expressed recombinant dromedary IFN-ß induced expression of IFN-responsive genes in camel kidney cells. Further, recombinant IFN-ß conferred antiviral resistance to camel kidney cells against the cytopathic effects of the camelpox virus, an endemic zoonotic pathogen. IFN-ß from this unique group of mammals will offer insights into antiviral immune mechanisms and aid in the development of specific antivirals against pathogens that have the potential to be the next zoonotic pandemic.

Serological evidence of SARS-CoV-2 infection in dromedary camels and domestic bovids in Oman.

SARS-CoV-2 has demonstrated the ability to infect a wide range of animal species. Here, we investigated SARS-CoV-2 infection in livestock species in Oman and provided serological evidence of SARS-CoV-2 infection in cattle, sheep, goats, and dromedary camel using the surrogate virus neutralization and plaque reduction neutralization tests. To better understand the extent of SARS-CoV-2 infection in animals and associated risks, "One Health" epidemiological investigations targeting animals exposed to COVID-19 human cases should be implemented with integrated data analysis of the epidemiologically linked human and animal cases.

MERS-CoV‒Specific T-Cell Responses in Camels after Single MVA-MERS-S Vaccination.

We developed an ELISPOT assay for evaluating Middle East respiratory syndrome coronavirus (MERS-CoV)‒specific T-cell responses in dromedary camels. After single modified vaccinia virus Ankara-MERS-S vaccination, seropositive camels showed increased levels of MERS-CoV‒specific T cells and antibodies, indicating suitability of camel vaccinations in disease-endemic areas as a promising approach to control infection.

MERS-CoV seroconversion amongst Malaysian Hajj pilgrims returning from the Middle East, 2016-2018: results from the MERCURIAL multiyear prospective cohort study.

Prospective cohort study to investigate the potential exposure to the Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) following Hajj pilgrims is still very limited. Here, we report the antibody seroconversion study results obtained from successive three years cohort studies (2016-2018) involving the Malaysian Hajj pilgrims returning from the Middle East. A cohort study of Hajj pilgrims from Malaysia enrolled 2,863 participants from 2016-2018, all of whom consented to provide paired blood samples for both pre- and post-Hajj travel to the Middle East. ELISAs and micro-neutralization assays were performed to detect the presence of MERS-CoV IgG antibodies. Sociodemographic data, symptoms experienced during Hajj, and history of exposure to camels or camel products were recorded using structured pre- and post-Hajj questionnaires. A 4-fold increase in anti-MERS-CoV IgG between paired pre-Hajj and post-Hajj serum samples in twelve participants was observed. None of the twelve ELISA-positive sera had detectable levels of virus-neutralizing antibodies. All reportedly had mild symptoms of respiratory symptoms at a certain point during the pilgrimage, implying mild or asymptomatic infections. No association between post-Hajj serum positivity and a history of exposure to camels or camel products was obtained. Findings from the study suggest that serologic conversion to MERS-CoV occurred in at least 0.6% of the Hajj pilgrims returning from the Middle East. Since all the seroconvertants had mild to no symptoms during the sampling period, it highlights the likelihood of occurrence of only low infectivity spillover infections among the Hajj pilgrims.

Detection of viral antibodies in camel sera using magnetic particle spectroscopy.

Pandemics like SARS-Cov-2 very frequently have their origin in different animals and in particular herds of camels could be a source of zoonotic diseases. This study took advantage on a highly sensitive and adaptable method for the fast and reliable detection of viral antibodies in camels using low-cost equipment. Magnetic nanoparticles (MNP) have high variability in their functionalization with different peptides and proteins. We confirm that 3-aminopropyl triethoxysilane (APTES)-coated MNP could be functionalized with viral proteins. The protein loading could be confirmed by simple loading controls using FACS-analysis (p < 0.05). Complementary combination of antigen and antibody yields in a significant signal increase could be proven by both FACS and COMPASS. However, COMPASS needs only a few seconds for the measurement. In COMPASS, the phase φn on selected critical point of the fifth higher harmonic (n = 5th). Here, positive sera display highly significant signal increase over the control or negative sera. Furthermore, a clear distinction could be made in antibody detection as an immune response to closely related viruses (SARS-CoV2 and MERS). Using modified MNPs along with COMPASS offers a fast and reliable method that is less cost intensive than current technologies and offers the possibility to be quickly adapted in case of new occurring viral infections. KEY POINTS: • COMPASS (critical offset magnetic particle spectroscopy) allows the fast detection of antibodies. • Magnetic nanoparticles can be adapted by exchange of the linked bait molecule. • Antibodies could be detected in camel sera without washing steps within seconds.

Extended Viral Shedding of MERS-CoV Clade B Virus in Llamas Compared with African Clade C Strain.

Middle East respiratory syndrome coronavirus (MERS-CoV) clade B viruses are found in camelids and humans in the Middle East, but clade C viruses are not. We provide experimental evidence for extended shedding of MERS-CoV clade B viruses in llamas, which might explain why they outcompete clade C strains in the Arabian Peninsula.

Middle East respiratory syndrome coronavirus-a 10-year (2012-2022) global analysis of human and camel infections, genomic sequences, lineages, and geographical origins.

OBJECTIVES: The World Health Organization priority zoonotic pathogen Middle East respiratory syndrome (MERS) coronavirus (CoV) has a high case fatality rate in humans and circulates in camels worldwide. METHODS: We performed a global analysis of human and camel MERS-CoV infections, epidemiology, genomic sequences, clades, lineages, and geographical origins for the period January 1, 2012 to August 3, 2022. MERS-CoV Surface gene sequences (4061 bp) were extracted from GenBank, and a phylogenetic maximum likelihood tree was constructed. RESULTS: As of August 2022, 2591 human MERS cases from 26 countries were reported to the World Health Organization (Saudi Arabia, 2184 cases, including 813 deaths [case fatality rate: 37.2%]) Although declining in numbers, MERS cases continue to be reported from the Middle East. A total of 728 MERS-CoV genomes were identified (the largest numbers were from Saudi Arabia [222: human = 146, camels = 76] and the United Arab Emirates [176: human = 21, camels = 155]). A total of 501 'S'-gene sequences were used for phylogenetic tree construction (camels [n = 264], humans [n = 226], bats [n = 8], other [n=3]). Three MERS-CoV clades were identified: clade B, which is the largest, followed by clade A and clade C. Of the 462 clade B lineages, lineage 5 was predominant (n = 177). CONCLUSION: MERS-CoV remains a threat to global health security. MERS-CoV variants continue circulating in humans and camels. The recombination rates indicate co-infections with different MERS-CoV lineages. Proactive surveillance of MERS-CoV infections and variants of concern in camels and humans worldwide, and development of a MERS vaccine, are essential for epidemic preparedness.

Genetic diversity and molecular epidemiology of Middle East Respiratory Syndrome Coronavirus in dromedaries in Ethiopia, 2017-2020.

Middle East respiratory syndrome coronavirus (MERS-CoV) is enzootic in dromedary camels and causes zoonotic infection and disease in humans. Although over 80% of the global population of infected dromedary camels are found in Africa, zoonotic disease had only been reported in the Arabia Peninsula and travel-associated disease has been reported elsewhere. In this study, genetic diversity and molecular epidemiology of MERS-CoV in dromedary camels in Ethiopia were investigated during 2017-2020. Of 1766 nasal swab samples collected, 61 (3.5%) were detected positive for MERS-CoV RNA. Of 484 turbinate swab samples collected, 10 (2.1%) were detected positive for MERS-CoV RNA. Twenty-five whole genome sequences were obtained from these MERS-CoV positive samples. Phylogenetically, these Ethiopian camel-originated MERS-CoV belonged to clade C2, clustering with other East African camel strains. Virus sequences from camel herds clustered geographically while in an abattoir, two distinct phylogenetic clusters of MERS-CoVs were observed in two sequential sampling collections, which indicates the greater genetic diversity of MERS-CoV in abattoirs. In contrast to clade A and B viruses from the Arabian Peninsula, clade C camel-originated MERS-CoV from Ethiopia had various nucleotide insertions and deletions in non-structural gene nsp3, accessory genes ORF3 and ORF5 and structural gene N. This study demonstrates the genetic instability of MERS-CoV in dromedaries in East Africa, which indicates that the virus is still actively adapting to its camel host. The impact of the observed nucleotide insertions and deletions on virus evolution, viral fitness, and zoonotic potential deserves further study.

Detection of Middle East Respiratory Syndrome Coronavirus-Specific RNA and Anti-MERS-Receptor-Binding Domain Antibodies in Camel Milk from Different Regions of Saudi Arabia.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes viral pneumonia disease in humans. The close contact with camels and drinking milk may cause MERS-CoV transfer to humans. This study was designed to detect the existence of MERS-CoV in camel milk samples collected from healthy animals of various barns located around Saudi Arabia. Camel milk samples were examined for MERS-CoV RNA by real time-quantitative polymerase chain reaction, also enzyme-linked immunosorbent assay was performed to detect IgG antibodies directed against Middle East respiratory syndrome receptor-binding domain. Among 83 camel milk samples tested, the result showed that seven samples (8.4%) were positive for MERS-CoV RNA, whereas 40.9% of camel milk samples had antibodies directed against MERS-CoV. The findings indicate that some regions (East and South part) are characterized by a high incidence of viral antibodies. The Southwestern region displayed the lowest infection rates. Among the camel breeds, the lowest positivity for detection of MERS-CoV RNA and IgG antibodies was found in Sahilia. This could be related to a higher resistance to viral infection of the breed Sahilia and/or to the geographical origin of the camels sampled in the study. This needs to be more explored to reduce spread of infection and also to understand the underlying reasons. The presence of viral RNA in camel milk samples warrants for measures to prevent possible foodborne transmission of MERS-CoV through milk consumption.

Novel type-I interferons from the dromedary camel: Molecular identification, prokaryotic expression and functional characterization of camelid interferon-delta.

The last two decades have seen the emergence of three highly pathogenic coronaviruses with zoonotic origins, which prompted immediate attention to the underlying cause and prevention of future outbreaks. Intensification of camel husbandry in the Middle East has resulted in increased human-camel interactions, which has led to the spread of potentially zoonotic viruses with human spillover risks like MERS-coronavirus, camelpox virus, etc. Type-I interferons function as the first line of defense against invading viruses and are pivotal for limiting viral replication and immune-mediated pathologies. Seven novel dromedary camel interferon delta genes were identified and cloned. Functional characterization of this novel class of IFNs from the mammalian suborder tylopoda is reported for the first time. The camel interferon-delta proteins resemble the reported mammalian counterparts in sequence similarity, conservation of cysteines, and phylogenetic proximity. Prokaryotically expressed recombinant camel interferon-δ1 induced IFN-stimulated gene expression and also exerted antiviral action against camelpox virus, an endemic zoonotic virus. The pre-treatment of camel kidney cells with recombinant camel IFN-δ1 increased cell survival and reduced camelpox virus in a dose-dependent manner. The identification of novel IFNs from species with zoonotic spillover risk such as camels, and evaluating their antiviral effects in-vitro will play a key role in improving immunotherapies against viruses and expanding the arsenal to combat emerging zoonotic pathogens.

Fractional model for Middle East respiratory syndrome coronavirus on a complex heterogeneous network.

In this paper, we present a new fractional epidemiological model on a heterogeneous network to investigate Middle East respiratory syndrome (MERS-CoV), which is caused by a virus in the coronavirus family. We also consider the development of equations for the camel population, given that it is the primary animal source of the virus, as well as direct human interaction with this population. The model is configured in an SIS form for both the human population and the camel population. We study the equilibrium positions of the system and the conditions for the existence of each of them, as well as the local stability of each equilibrium position. Then, we provide some numerical examples that compare real data and numerical results.

Screening, Expression, and Identification of Nanobody against SARS-CoV-2 Spike Protein.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an infectious disease that has become a serious burden on global public health. This study screened and yielded specific nanobodies (Nbs) against SARS-CoV-2 spike protein receptor binding domain (RBD), following testing its basic characteristics. A nanobody phage library was established by immunizing a camel with RBD protein. After three rounds of panning, the positive colonies were screened by enzyme-linked immunosorbent assay (ELISA). By sequencing, four different sequences of nanobody gene fragments were selected. The four nanobody fusion proteins were expressed and purified, respectively. The specificity and affinity of the four nanobodies were identified by ELISA. Our results showed that an immune phage display library against SARS-CoV-2 has been successfully constructed with a library capacity of which was 4.7 × 108 CFU. The four purified nanobodies showed specific high-affinity binding SARS-CoV-2 S-RBD. Among these, the antigen binding affinity of Nb61 was more comparable to that of commercial rabbit anti-SARS-CoV-2 S-RBD antibodies. In sum, our study has obtained four nanobody strains against SARS-CoV-2 S-RBD with significant affinity and specificity, therefore laying an essential foundation for further research as well as the applications of diagnostic and therapeutic tools of SARS-CoV-2.

A megadiverse naïve library derived from numerous camelids for efficient and rapid development of VHH antibodies.

The field of antibody development is under pressure to meet rising demands for speed, cost-effectiveness, efficacy, reliability, and large-scale production. It is costly and time-consuming to immunize animals and build a single-domain antibody (sdAb) library for each target. Using the variable domain (VHH) of heavy-chain only antibodies (HcAbs) derived from blood samples of 75 non-immunized camelid animals (51 alpacas, 13 llamas, 11 Bactrian camels), and spleens from two Bactrian camels, a naïve sdAb library with extensive megadiversity and reusability was constructed. The library was evaluated using next-generation DNA sequencing (NGS) and was found to contain hundreds of billions of unique clones. To confirm the availability of target-specific VHHs, a naive library was screened for a variety of targets. At least two VHH candidates were extracted for each target using a 20-day selection pipeline. Some binders had ultrahigh potencies, with binding affinities in the nanomolar range. This naïve library, in particular, offers the possibility of acquiring unique antibodies targeting antigens of interest with low feasible dissociation constant (kD) without the time, effort, and price associated in producing antibodies in animals via antigen injection. Overall, the study shows that the megadiverse naïve library provides a rapid, adaptable, and easy platform for antibody creation, emphasizing its therapeutic and diagnostic implications.

Ecology of Middle East respiratory syndrome coronavirus, 2012-2020: A machine learning modelling analysis.

The ongoing enzootic circulation of the Middle East respiratory syndrome coronavirus (MERS-CoV) in the Middle East and North Africa is increasingly raising the concern about the possibility of its recombination with other human-adapted coronaviruses, particularly the pandemic SARS-CoV-2. We aim to provide an updated picture about ecological niches of MERS-CoV and associated socio-environmental drivers. Based on 356 confirmed MERS cases with animal contact reported to the WHO and 63 records of animal infections collected from the literature as of 30 May 2020, we assessed ecological niches of MERS-CoV using an ensemble model integrating three machine learning algorithms. With a high predictive accuracy (area under receiver operating characteristic curve = 91.66% in test data), the ensemble model estimated that ecologically suitable areas span over the Middle East, South Asia and the whole North Africa, much wider than the range of reported locally infected MERS cases and test-positive animal samples. Ecological suitability for MERS-CoV was significantly associated with high levels of bareland coverage (relative contribution = 30.06%), population density (7.28%), average temperature (6.48%) and camel density (6.20%). Future surveillance and intervention programs should target the high-risk populations and regions informed by updated quantitative analyses.

MERS-CoV ORF4b is a virulence factor involved in the inflammatory pathology induced in the lungs of mice.

No vaccines or specific antiviral drugs are authorized against Middle East respiratory syndrome coronavirus (MERS-CoV) despite its high mortality rate and prevalence in dromedary camels. Since 2012, MERS-CoV has been causing sporadic zoonotic infections in humans, which poses a risk of genetic evolution to become a pandemic virus. MERS-CoV genome encodes five accessory proteins, 3, 4a, 4b, 5 and 8b for which limited information is available in the context of infection. This work describes 4b as a virulence factor in vivo, since the deletion mutant of a mouse-adapted MERS-CoV-Δ4b (MERS-CoV-MA-Δ4b) was completely attenuated in a humanized DPP4 knock-in mouse model, resulting in no mortality. Attenuation in the absence of 4b was associated with a significant reduction in lung pathology and chemokine expression levels at 4 and 6 days post-infection, suggesting that 4b contributed to the induction of lung inflammatory pathology. The accumulation of 4b in the nucleus in vivo was not relevant to virulence, since deletion of its nuclear localization signal led to 100% mortality. Interestingly, the presence of 4b protein was found to regulate autophagy in the lungs of mice, leading to upregulation of BECN1, ATG3 and LC3A mRNA. Further analysis in MRC-5 cell line showed that, in the context of infection, MERS-CoV-MA 4b inhibited autophagy, as confirmed by the increase of p62 and the decrease of ULK1 protein levels, either by direct or indirect mechanisms. Together, these results correlated autophagy activation in the absence of 4b with downregulation of a pathogenic inflammatory response, thus contributing to attenuation of MERS-CoV-MA-Δ4b.

Cross-sectional study of Middle East respiratory syndrome coronavirus in humans and dromedary camels in Diyala, Iraq.

BACKGROUND: Middle East respiratory syndrome (MERS) is a zoonotic viral disease transmitted from dromedaries to humans. To date, more than 1500 cases of MERS have been reported and 80% of all cases have occurred in the Kingdom of Saudi Arabia (KSA). This cross-sectional study was carried out to figure out the rate of infection among humans and dromedary camels and to explore the risk factors. METHODS: This study was conducted in Diyala Province, Iraq for the period from August 2017 to October 2018. Human subjects included 90 participants; 34 (37.8%) were females and 56 (62.2%) were males. Additionally, 90 dromedary camels were also included, 50 (55.6%) males and 40 (44.4%) females. Serum samples from subjects were collected and tested for the presence of anti-MERS-coronavirus (CoV) immunoglobulin g (IgG). RESULTS: The results revealed that 46 (51.1%) of human subjects were positive for anti-MERS-CoV IgG, (95% confidence interval (CI) for the prevalence rate 40.9-61.3) with a mean titer of anti-MERS-CoV IgG antibodies (Ab) of 81.2 U/mL. The anti-MERS-CoV IgG positivity rate was insignificantly higher, but the mean of anti-MERS-CoV IgG titer was significantly higher among females (p = 0.12 and p < 0.004, respectively). Furthermore, the anti-MERS-CoV IgG positivity rate and Ab titer were significantly higher among those people who visited KSA for Hajj or Umrah (p < 0.001 and p < 0.001, respectively). In camels, 81 (90.0%) were positive for anti-MERS-CoV IgG, (95% CI for the prevalence rate 82.5-94.9), with a mean titer of 99.8 U/mL. DISCUSSION: The MERS-CoV infection rate was high among both Iraqi humans and dromedary camels. Further confirmatory studies are needed, and setting up of national precaution program is essential.

Outbreak of Middle East Respiratory Syndrome Coronavirus in Camels and Probable Spillover Infection to Humans in Kenya.

The majority of Kenya's > 3 million camels have antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV), although human infection in Africa is rare. We enrolled 243 camels aged 0-24 months from 33 homesteads in Northern Kenya and followed them between April 2018 to March 2020. We collected and tested camel nasal swabs for MERS-CoV RNA by RT-PCR followed by virus isolation and whole genome sequencing of positive samples. We also documented illnesses (respiratory or other) among the camels. Human camel handlers were also swabbed, screened for respiratory signs, and samples were tested for MERS-CoV by RT-PCR. We recorded 68 illnesses among 58 camels, of which 76.5% (52/68) were respiratory signs and the majority of illnesses (73.5% or 50/68) were recorded in 2019. Overall, 124/4692 (2.6%) camel swabs collected from 83 (34.2%) calves in 15 (45.5%) homesteads between April-September 2019 screened positive, while 22 calves (26.5%) recorded reinfections (second positive swab following ≥ 2 consecutive negative tests). Sequencing revealed a distinct Clade C2 virus that lacked the signature ORF4b deletions of other Clade C viruses. Three previously reported human PCR positive cases clustered with the camel infections in time and place, strongly suggesting sporadic transmission to humans during intense camel outbreaks in Northern Kenya.

Camel milk consumption patterns and perceptions in the UAE: a cross-sectional study.

Camel milk has been consumed for centuries due to its medicinal and healing properties. The present study aims to investigate the consumption patterns of camel milk and perceived benefits and risks among adults in the United Arab Emirates. A self-administered online questionnaire was developed in English and Arabic languages and was completed online by 852 adults. Socio-demographic characteristics, camel milk consumption patterns and perceived knowledge of the benefits and risks of camel milk were investigated. About 60 % of the participants have tried drinking camel milk, but only a quarter (25⋅1 %) were regular consumers. The most consumed camel milk products after fresh milk were yoghurt and flavoured milk. The most popular additions to camel milk were honey, turmeric and sugar. Most consumers had less than one cup of camel milk per day (57⋅0 %). Camel milk consumers preferred it over other types of milk due to its nutritional value (66⋅4 %) and medicinal properties (39⋅3 %). Among consumers, 58⋅4 % reported consuming unpasteurised camel milk. Reasons included the belief that it is fresher (87⋅2 %), better for the immune system (41⋅6 %), and higher in nutrients (39⋅2 %). Overall, participants had inadequate knowledge about the health benefits of camel milk (7⋅11 ± 5⋅3 out of 25). Males and camel milk consumers had a significantly higher knowledge about the health benefits of camel milk compared to females and non-consumers (P < 0⋅05). Although positive perceptions were common, misperceptions appear to be prevalent among non-consumers. Consuming unpasteurised camel milk is a major public health concern, thus national regulations are essential.