Comparative pathogenic potential of avian influenza H7N3 viruses isolated from wild birds in Egypt and their sensitivity to commercial antiviral drugs.

Active surveillance and studying the virological features of avian-origin influenza viruses are essential for early warning and preparedness for the next potential pandemic. During our active surveillance of avian influenza viruses in wild birds in Egypt in the period 2014-2017, multiple reassortant low-pathogenic avian influenza H7N3 viruses were isolated. In this study, we investigated and compared the infectivity, pathogenicity, and transmission of four different constellation forms of Egyptian H7N3 viruses in chickens and mice and assessed the sensitivity of these viruses to different commercial antiviral drugs in vitro. Considerable variation in virus pathogenicity was observed in mice infected with different H7N3 viruses. The mortality rate ranged from 20 to 100% in infected mice. Infected chickens showed only ocular clinical signs at three days postinfection as well as systemic viral infection in different organs. Efficient virus replication and transmission in chickens was observed within each group, indicating that these subtypes can spread easily from wild birds to poultry without prior adaptation. Mutations in the viral proteins associated with antiviral drug resistance were not detected, and all strains were sensitive to the antiviral drugs tested. In conclusion, all of the viruses studied had the ability to infect mice and chickens. H7N3 viruses circulating among wild birds in Egypt could threaten poultry production and public health.

Molecular identification and virological characteristics of highly pathogenic avian influenza A/H5N5 virus in wild birds in Egypt.

Multiple incursions of different subtypes of highly pathogenic avian influenza (HPAI) A/H5NX viruses have caused widely considerable outbreaks in poultry and hundreds of human infections. Extensive reassortment events associated with currently circulating clade 2.3.4.4b of A/H5NX viruses have been widely recorded. Wild migratory birds contribute to the spillover of diverse viruses throughout their migration flyways. During our active surveillance of avian influenza in Egypt, we successfully isolated and fully characterized HPAI A/H5N5 virus of clade 2.3.4.4b that was detected in a healthy purple heron. The Egyptian H5N5 virus is genotypically similar with the same subtype that was detected in the far east of Russia and several European countries. The antigenic analysis showed that the Egyptian H5N5 virus is distinct from HPAI A(H5N8) viruses in Egypt. The virus preferentially binds to avian-like receptors rather than human-like receptors. Our results showed that the virus caused 100% and 60% lethality in chicken and mice respectively. Increasing active surveillance efforts, monitoring the dynamics of emerging AIVs, and risk assessment implementation should be globally applied especially in hot spot regions like Egypt.

Bioactive Polyphenolic Compounds Showing Strong Antiviral Activities against Severe Acute Respiratory Syndrome Coronavirus 2.

Until now, there has been no direct evidence of the effectiveness of repurposed FDA-approved drugs against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections. Although curcumin, hesperidin, and quercetin have broad spectra of pharmacological properties, their antiviral activities against SARS-CoV-2 remain unclear. Our study aimed to assess the in vitro antiviral activities of curcumin, hesperidin, and quercetin against SARS-CoV-2 compared to hydroxychloroquine and determine their mode of action. In Vero E6 cells, these compounds significantly inhibited virus replication, mainly as virucidal agents primarily indicating their potential activity at the early stage of viral infection. To investigate the mechanism of action of the tested compounds, molecular docking studies were carried out against both SARS-CoV-2 spike (S) and main protease (Mpro) receptors. Collectively, the obtained in silico and in vitro findings suggest that the compounds could be promising SARS-CoV-2 Mpro inhibitors. We recommend further preclinical and clinical studies on the studied compounds to find a potential therapeutic targeting COVID-19 in the near future.

Immunogenicity and Safety of an Inactivated SARS-CoV-2 Vaccine: Preclinical Studies.

Since the emergence of SARS-CoV-2 at the end of 2019, 64 candidate vaccines are in clinical development and 173 are in the pre-clinical phase. Five types of vaccines are currently approved for emergency use in many countries (Inactivated, Sinopharm; Viral-vector, Astrazeneca, and Gamaleya Research Institute; mRNA, Moderna, and BioNTech/Pfizer). The main challenge in this pandemic was the availability to produce an effective vaccine to be distributed to the world's population in a short time. Herein, we developed a whole virus NRC-VACC-01 inactivated candidate SARS-CoV-2 vaccine and tested its safety and immunogenicity in laboratory animals. In the preclinical studies, we used four experimental animals (mice, rats, guinea pigs, and hamsters). Antibodies were detected as of week three post vaccination and continued up to week ten in the four experimental models. Safety evaluation of NRC-VACC-01 inactivated candidate vaccine in rats revealed that the vaccine was highly tolerable. By studying the effect of booster dose in the immunological profile of vaccinated mice, we observed an increase in neutralizing antibody titers after the booster shot, thus a booster dose was highly recommended after week three or four. Challenge infection of hamsters showed that the vaccinated group had lower morbidity and shedding than the control group. A phase I clinical trial will be performed to assess safety in human subjects.

Molecular Characterization of Closely Related H6N2 Avian Influenza Viruses Isolated from Turkey, Egypt, and Uganda.

Genetic analysis of circulating avian influenza viruses (AIVs) in wild birds at different geographical regions during the same period could improve our knowledge about virus transmission dynamics in natural hosts, virus evolution as well as zoonotic potential. Here, we report the genetic and molecular characterization of H6N2 influenza viruses isolated from migratory birds in Turkey, Egypt, and Uganda during 2017-2018. The Egyptian and Turkish isolates were genetically closer to each other than they were to the virus isolated from Uganda. Our results also suggest that multiple reassortment events were involved in the genesis of the isolated viruses. All viruses contained molecular markers previously associated with increased replication and/or pathogenicity in mammals. The results of this study indicate that H6N2 viruses carried by migratory birds on the West Asian/East African and Mediterranean/Black Sea flyways have the potential to transmit to mammals including humans. Additionally, adaptation markers in these viruses indicate the potential risk for poultry, which also increases the possibility of human exposure to these viruses.

Incidence, household transmission, and neutralizing antibody seroprevalence of Coronavirus Disease 2019 in Egypt: Results of a community-based cohort.

SARS-CoV-2 virus is transmitted in closed settings to people in contact with COVID-19 patients such as healthcare workers and household contacts. However, household person-to-person transmission studies are limited. Households participating in an ongoing cohort study of influenza incidence and prevalence in rural Egypt were followed. Baseline enrollment was done from August 2015 to March 2017. The study protocol was amended in April 2020 to allow COVID-19 incidence and seroprevalence studies. A total of 290 households including 1598 participants were enrolled and followed from April to October 2020 in four study sites. When a participant showed respiratory illness symptoms, a serum sample and a nasal and an oropharyngeal swab were obtained. Swabs were tested by RT-PCR for SARS-CoV-2 infection. If positive, the subject was followed and swabs collected on days three, six, nine, and 14 after the first swab day and a serum sample obtained on day 14. All subjects residing with the index case were swabbed following the same sampling schedule. Sera were collected from cohort participants in October 2020 to assess seroprevalence. Swabs were tested by RT-PCR. Sera were tested by Microneutralization Assay to measure the neutralizing antibody titer. Incidence of COVID-19, household secondary attack rate, and seroprevalence in the cohort were determined. The incidence of COVID-19 was 6.9% and the household secondary attack rate was 89.8%. Transmission within households occurred within two-days of confirming the index case. Infections were asymptomatic or mild with symptoms resolving within 10 days. The majority developed a neutralizing antibody titer by day 14 post onset. The overall seroprevalence among cohort participants was 34.8%. These results suggest that within-household transmission is high in Egypt. Asymptomatic or mild illness is common. Most infections seroconvert and have a durable neutralizing antibody titer.

FDA-Approved Drugs with Potent In Vitro Antiviral Activity against Severe Acute Respiratory Syndrome Coronavirus 2.

(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.

Prevalence of Severe Acute Respiratory Syndrome Coronavirus 2 Neutralizing Antibodies in Egyptian Convalescent Plasma Donors.

Using convalescent plasma as immunotherapy is an old method for treatment of infectious diseases. Several countries have recently allowed the use of such therapy for the treatment of COVID-19 patients especially those who are critically ill. A similar program is currently being tested in Egypt. Here, we tested 227 plasma samples from convalescent donors in Egypt for neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using a microneutralization (MN) assay. A third of the tested samples did not have antibody titers and 58% had titers between 1:10 and 1:80. Only 12% had titers >1:160. We also compared MN assays using different virus concentrations, plaque reduction neutralization (PRNT) assays, and a chemiluminescence assay that measures immunoglobulin G (IgG) binding to N and S proteins of SARS-CoV-2. Our results indicated that a MN assay using 100 TCID50/ml provides comparable results to PRNT and allows for high throughput testing.

Common childhood vaccines do not elicit a cross-reactive antibody response against SARS-CoV-2.

Anecdotal evidence showed a negative correlation between Bacille Calmette-Guérin (BCG) vaccination and incidence of COVID-19. Incidence of the disease in children is much lower than in adults. It is hypothesized that BCG and other childhood vaccinations may provide some protection against SARS-CoV-2 infection through trained or adaptive immune responses. Here, we tested whether BCG, Pneumococcal, Rotavirus, Diphtheria, Tetanus, Pertussis, Hepatitis B, Haemophilus influenzae, Hepatitis B, Meningococcal, Measles, Mumps, and Rubella vaccines provide cross-reactive neutralizing antibodies against SARS-CoV-2 in BALB/c mice. Results indicated that none of these vaccines provided antibodies capable of neutralizing SARS-CoV-2 up to seven weeks post vaccination. We conclude that if such vaccines have any role in COVID-19 immunity, this role is not antibody-mediated.

Coding-Complete Genome Sequences of Two SARS-CoV-2 Isolates from Egypt.

This report announces the complete genome sequences of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolates detected in Egypt. The isolates were obtained from oropharyngeal swab specimens from two Egyptians in Upper and Lower Egypt. Sequence analysis showed mutations that differentiate Egyptian strains from the reference strain 2019-nCoV WHU01.

Active surveillance and genetic evolution of avian influenza viruses in Egypt, 2016-2018.

Egypt is a hotspot for avian influenza virus (AIV) due to the endemicity of H5N1 and H9N2 viruses. AIVs were isolated from 329 samples collected in 2016-2018; 48% were H9N2, 37.1% were H5N8, 7.6% were H5N1, and 7.3% were co-infections with 2 of the 3 subtypes. The 32 hemagglutinin (HA) sequences of the H5N1 viruses formed a well-defined lineage within clade 2.2.1.2. The 10 HA sequences of the H5N8 viruses belonged to a subclade within 2.3.4.4. The 11 HA of H9N2 isolates showed high sequence homology with other Egyptian G1-like H9N2 viruses. The prevalence of H5N8 viruses in ducks (2.4%) was higher than in chickens (0.94%). Genetic reassortment was detected in H9N2 viruses. Antigenic analysis showed that H9N2 viruses are homogenous, antigenic drift was detected among H5N1 viruses. AI H5N8 showed higher replication rate followed by H9N2 and H5N1, respectively. H5N8 was more common in Southern Egypt, H9N2 in the Nile Delta, and H5N1 in both areas. Ducks and chickens played a significant role in transmission of H5N1 viruses. The endemicity and co-circulation of H5N1, H5N8, and H9N2 AIV coupled with the lack of a clear control strategy continues to provide avenues for further virus evolution in Egypt.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Dromedary Camels in Africa and Middle East.

: Dromedary camels are the natural reservoirs of the Middle East respiratory syndrome coronavirus (MERS-CoV). Camels are mostly bred in East African countries then exported into Africa and Middle East for consumption. To understand the distribution of MERS-CoV among camels in North Africa and the Middle East, we conducted surveillance in Egypt, Senegal, Tunisia, Uganda, Jordan, Saudi Arabia, and Iraq. We also performed longitudinal studies of three camel herds in Egypt and Jordan to elucidate MERS-CoV infection and transmission. Between 2016 and 2018, a total of 4027 nasal swabs and 3267 serum samples were collected from all countries. Real- time PCR revealed that MERS-CoV RNA was detected in nasal swab samples from Egypt, Senegal, Tunisia, and Saudi Arabia. Microneutralization assay showed that antibodies were detected in all countries. Positive PCR samples were partially sequenced, and a phylogenetic tree was built. The tree suggested that all sequences are of clade C and sequences from camels in Egypt formed a separate group from previously published sequences. Longitudinal studies showed high seroprevalence in adult camels. These results indicate the widespread distribution of the virus in camels. A systematic active surveillance and longitudinal studies for MERS-CoV are needed to understand the epidemiology of the disease and dynamics of viral infection.

Development of an effective contemporary trivalent avian influenza vaccine against circulating H5N1, H5N8, and H9N2 in Egypt.

Low pathogenicity avian influenza (LPAI) H9N2, highly pathogenic avian influenza (HPAI) H5N1, and H5N8 circulate in Egyptian poultry and cause veterinary and public health burdens. In response, AIV vaccines are commonly used. The main objective of this study was to develop a broad, cross-protective, trivalent vaccine based on circulating AIVs in Egypt. We generated highly replicating avirulent AIVs, H5N1, and H5N8, to be used in combination with H9N2 strain for the generation of an inactivated vaccine. Immunogenicity and protective efficacy of this vaccine were tested. Results showed that a single immunization dose enhanced humoral immune responses giving full protection against challenges with LPAI H9N2, HPAI H5N1, and H5N8 viruses. This efficacious vaccine will reduce the cost of vaccination for poultry growers and is expected to be effective in the field as it is based on contemporary viruses currently in circulation among Egyptian poultry.

Middle East respiratory syndrome coronavirus infection in non-camelid domestic mammals.

Dromedary camels are natural host of the Middle East respiratory syndrome coronavirus (MERS-CoV). However, there are limited studies of MERS-CoV infection of other domestic mammals exposed to infected dromedaries. We expanded our surveillance among camels in Egypt, Tunisia, and Senegal to include other domestic mammalian species in contact with infected camels. A total of 820 sera and 823 nasal swabs from cattle, sheep, goats, donkeys, buffaloes, mules, and horses were collected. Swabs were tested using RT-PCR and virus RNA-positive samples were genetically sequenced and phylogenetically analysed. Sera were screened using virus microneutralization tests and positive sera (where available) were confirmed using plaque reduction neutralization tests (PRNT). We detected 90% PRNT confirmed MERS-CoV antibody in 35 (55.6%) of 63 sera from sheep collected from Senegal, two sheep (1.8%) of 114 in Tunisia and a goat (0.9%) of 107 in Egypt, with titres ranging from 1:80 to ≥1:320. We detected MERS-CoV RNA in swabs from three sheep (1.2%) of 254 and five goats (4.1%) of 121 from Egypt and Senegal, as well as one cow (1.9%) of 53 and three donkeys (7.1%) of 42 from Egypt. Partial sequences of the RT-PCR amplicons confirmed specificity of the results. This study showed that domestic livestock in contact with MERS-CoV infected camels may be at risk of infection. We recommend expanding current MERS-CoV surveillance in animals to include other livestock in close contact with dromedary camels. The segregation of camels from other livestock in farms and live animal markets may need to be considered.

Surveillance for avian influenza viruses in wild birds at live bird markets, Egypt, 2014-2016.

AIM: Egypt is the habitat for a large number of bird species and serves as a vital stopover for millions of migratory birds during their annual migration between the Palearctic and Afrotropical ecozones. Surveillance for avian influenza viruses (AIVs) is critical to assessing risks for potential spreading of these viruses among domestic poultry. Surveillance for AIV among hunted and captured wild birds in Egypt was conducted in order to understand the characteristics of circulating viruses. METHODS: Sampling of wild bird species occurred in two locations along the Mediterranean Coast of Egypt in the period from 2014 to 2016. A total of 1316 samples (cloacal and oropharyngeal swabs) were collected from 20 different species of hunted or captured resident and migratory birds sold at live bird markets. Viruses were propagated then sequenced. Phylogenetic analysis and receptor binding affinities were studied. RESULTS: Eighteen AIVs (1.37%) were isolated from migratory Anseriformes at live bird markets. Further characterization of the viral isolates identified five hemagglutinin (H3, H5, H7, H9, and H10) and five neuraminidase (N1, N2, N3, N6, and N9) subtypes, which were related to isolates reported in the Eurasian region. Two of the 18 isolates were highly pathogenic H5N1 viruses related to clade 2.2.1, while three isolates were G1-like H9N2 viruses. CONCLUSIONS: Our data show significant diversity of AIVs in Anserifromes sold at live bird markets in Egypt. This allows for genetic exchanges between imported and enzootic viruses and put the exposed humans at a higher risk of infection.

Isolation and Characterization of a Distinct Influenza A Virus from Egyptian Bats.

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.

Genetic characterization of highly pathogenic avian influenza A H5N8 viruses isolated from wild birds in Egypt.

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.

Systematic, active surveillance for Middle East respiratory syndrome coronavirus in camels in Egypt.

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.

Serological Evidence of Human Infection with Avian Influenza A H7virus in Egyptian Poultry Growers.

Avian influenza viruses circulate widely in birds, with occasional human infections. Poultry-exposed individuals are considered to be at high risk of infection with avian influenza viruses due to frequent exposure to poultry. Some avian H7 viruses have occasionally been found to infect humans. Seroprevalence of neutralizing antibodies against influenza A/H7N7 virus among poultry-exposed and unexposed individuals in Egypt were assessed during a three-years prospective cohort study. The seroprevalence of antibodies (titer, ≥80) among exposed individuals was 0%, 1.9%, and 2.1% annually while the seroprevalence among the control group remained 0% as measured by virus microneutralization assay. We then confirmed our results using western blot and immunofluorescence assays. Although human infection with H7 in Egypt has not been reported yet, our results suggested that Egyptian poultry growers are exposed to avian H7 viruses. These findings highlight the need for surveillance in the people exposed to poultry to monitor the risk of zoonotic transmission of avian influenza viruses.

Avian Influenza A(H5N1) Virus in Egypt.

In Egypt, avian influenza A subtype H5N1 and H9N2 viruses are enzootic in poultry. The control plan devised by veterinary authorities in Egypt to prevent infections in poultry focused mainly on vaccination and ultimately failed. Recently, widespread H5N1 infections in poultry and a substantial increase in the number of human cases of H5N1 infection were observed. We summarize surveillance data from 2009 through 2014 and show that avian influenza viruses are established in poultry in Egypt and are continuously evolving genetically and antigenically. We also discuss the epidemiology of human infection with avian influenza in Egypt and describe how the true burden of disease is underestimated. We discuss the failures of relying on vaccinating poultry as the sole intervention tool. We conclude by highlighting the key components that need to be included in a new strategy to control avian influenza infections in poultry and humans in Egypt.