We describe a novel spike pseudoparticle neutralisation assay (ppNT) for seroepidemiological studies on Middle East respiratory syndrome coronavirus (MERS-CoV) and apply this assay together with conventional microneutralisation (MN) tests to investigate 1,343 human and 625 animal sera. The sera were collected in Egypt as a region adjacent to areas where MERS has been described, and in Hong Kong, China as a control region. Sera from dromedary camels had a high prevalence of antibody reactive to MERS-CoV by MERS NT (93.6%) and MERS ppNT (98.2%) assay. The antibody titres ranged up to 1,280 and higher in MN assays and 10,240 and higher in ppNT assays. No other investigated species had any antibody reactivity to MERS-CoV. While seropositivity does not exclude the possibility of infection with a closely related virus, our data highlight the need to attempt detection of MERS-CoV or related coronaviruses in dromedary camels. The data show excellent correlation between the conventional MN assay and the novel ppNT assay. The newly developed ppNT assay does not require Biosafety Level 3 containment and is thus a relatively high-throughput assay, well suited for large-scale seroepidemiology studies which are needed to better understand the ecology and epidemiology of MERS-CoV.

(1) Background: Drug repositioning is an unconventional drug discovery approach to explore new therapeutic benefits of existing drugs. Currently, it emerges as a rapid avenue to alleviate the COVID-19 pandemic disease. (2) Methods: Herein, we tested the antiviral activity of anti-microbial and anti-inflammatory Food and Drug Administration (FDA)-approved drugs, commonly prescribed to relieve respiratory symptoms, against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the viral causative agent of the COVID-19 pandemic. (3) Results: Of these FDA-approved antimicrobial drugs, Azithromycin, Niclosamide, and Nitazoxanide showed a promising ability to hinder the replication of a SARS-CoV-2 isolate, with IC50 of 0.32, 0.16, and 1.29 µM, respectively. We provided evidence that several antihistamine and anti-inflammatory drugs could partially reduce SARS-CoV-2 replication in vitro. Furthermore, this study showed that Azithromycin can selectively impair SARS-CoV-2 replication, but not the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). A virtual screening study illustrated that Azithromycin, Niclosamide, and Nitazoxanide bind to the main protease of SARS-CoV-2 (Protein data bank (PDB) ID: 6lu7) in binding mode similar to the reported co-crystalized ligand. Also, Niclosamide displayed hydrogen bond (HB) interaction with the key peptide moiety GLN: 493A of the spike glycoprotein active site. (4) Conclusions: The results suggest that Piroxicam should be prescribed in combination with Azithromycin for COVID-19 patients.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe human infections and dromedary camels are considered an intermediary host. The dynamics of natural infection in camels are not well understood. Through systematic surveillance in Egypt, nasal, rectal, milk, urine and serum samples were collected from camels between June 2014 and February 2016. Locations included quarantines, markets, abattoirs, free-roaming herds and farmed breeding herds. The overall seroprevalence was 71% and RNA detection rate was 15%. Imported camels had higher seroprevalence (90% vs 61%) and higher RT-PCR detection rates (21% vs 12%) than locally raised camels. Juveniles had lower seroprevalence than adults (37% vs 82%) but similar RT-PCR detection rates (16% vs 15%). An outbreak in a breeding herd, showed that antibodies rapidly wane, that camels become re-infected, and that outbreaks in a herd are sustained for an extended time. Maternal antibodies titers were very low in calves regardless of the antibody titers of the mothers. Our results support the hypothesis that camels are a reservoir for MERS-CoV and that camel trade is an important route of introducing the virus into importing countries. Findings related to waning antibodies and re-infection have implications for camel vaccine development, disease management and zoonotic threat.

Continuous circulation of influenza A(H5N1) virus among poultry in Egypt has created an epicenter in which the viruses evolve into newer subclades and continue to cause disease in humans. To detect influenza viruses in Egypt, since 2009 we have actively surveyed various regions and poultry production sectors. From August 2010 through January 2013, >11,000 swab samples were collected; 10% were positive by matrix gene reverse transcription PCR. During this period, subtype H9N2 viruses emerged, cocirculated with subtype H5N1 viruses, and frequently co-infected the same avian host. Genetic and antigenic analyses of viruses revealed that influenza A(H5N1) clade 2.2.1 viruses are dominant and that all subtype H9N2 viruses are G1-like. Cocirculation of different subtypes poses concern for potential reassortment. Avian influenza continues to threaten public and animal health in Egypt, and continuous surveillance for avian influenza virus is needed.

Avian influenza virus subtype H9N2 has been circulating in the Middle East since the 1990s. For uncertain reasons, H9N2 was not detected in Egyptian farms until the end of 2010. Circulation of H9N2 viruses in Egyptian poultry in the presence of the enzootic highly pathogenic H5N1 subtype adds a huge risk factor to the Egyptian poultry industry. In this study, 22 H9N2 viruses collected from 2011 to 2013 in Egypt were isolated and sequenced. The genomic signatures and protein sequences of these isolates were analyzed. Multiple mammalian-host-associated mutations were detected that favor transmission from avian to mammalian hosts. Other mutations related to virulence were also identified. Phylogenetic data showed that Egyptian H9N2 viruses were closely related to viruses isolated from neighboring Middle Eastern countries, and their HA gene resembled those of viruses of the G1-like lineage. No reassortment was detected with H5N1 subtypes. Serological analysis of H9N2 virus revealed antigenic conservation among Egyptian isolates. Accordingly, continuous surveillance that results in genetic and antigenic characterization of H9N2 in Egypt is warranted.

A newly emerged H5N8 influenza virus was isolated from green-winged teal in Egypt during December 2016. In this study, we provide a detailed characterization of full genomes of Egyptian H5N8 viruses and some virological features. Genetic analysis demonstrated that the Egyptian H5N8 viruses are highly pathogenic avian influenza viruses. Phylogenetic analysis revealed that the genome of the Egyptian H5N8 viruses was related to recently characterized reassortant H5N8 viruses of clade 2.3.4.4 isolated from different Eurasian countries. Multiple peculiar mutations were characterized in the Egyptian H5N8 viruses, which probably permits transmission and virulence of these viruses in mammals. The Egyptian H5N8 viruses preferentially bound to avian-like receptors rather than human-like receptors. Also, the Egyptian H5N8 viruses were fully sensitive to amantadine and neuraminidase inhibitors. Chicken sera raised against commercial inactivated avian influenza-H5 vaccines showed no or very low reactivity with the currently characterized H5N8 viruses in agreement with the genetic dissimilarity. Surveillance of avian influenza in waterfowl provides early warning of specific threats to poultry and human health and hence should be continued.

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Recently, two genetically distinct influenza viruses were detected in bats in Guatemala and Peru. We conducted influenza A virus surveillance among four bat species in Egypt. Out of 1,202 swab specimens, 105 were positive by real-time PCR. A virus was successfully isolated in eggs and propagated in MDCK cells in the presence of N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin. Genomic analysis revealed that the virus was phylogenetically distinct from all other influenza A viruses. Analysis of the hemagglutinin gene suggested a common ancestry with other H9 viruses, and the virus showed a low level of cross-reactivity with serum raised against H9N2 viruses. Bats were seropositive for the isolated viruses. The virus replicated in the lungs of experimentally infected mice. While it is genetically distinct, this virus shares several avian influenza virus characteristics suggesting a more recent avian host origin. IMPORTANCE Through surveillance, we isolated and characterized an influenza A virus from Egyptian fruit bats. This virus had an affinity to avian-like receptors but was also able to infect mice. Our findings indicate that bats may harbor a diversity of influenza A viruses. Such viruses may have the potential to cross the species barrier to infect other species, including domestic birds, mammals, and, possibly, humans.