Genetic and virological characteristics of a reassortant avian influenza A H6N1 virus isolated from wild birds at a live-bird market in Egypt.

The first detection of a human infection with avian influenza A/H6N1 virus in Taiwan in 2013 has raised concerns about this virus. During our routine surveillance of avian influenza viruses (AIVs) in live-bird markets in Egypt, an H6N1 virus was isolated from a garganey duck and was characterized. Phylogenetic analysis indicated that the Egyptian H6N1 strain A/Garganey/Egypt/20869C/2022(H6N1) has a unique genomic constellation, with gene segments inherited from different subtypes (H5N1, H3N8, H7N3, H6N1, and H10N1) that have been detected previously in AIVs from Egypt and some Eurasian countries. We examined the replication of kinetics of this virus in different mammalian cell lines (A549, MDCK, and Vero cells) and compared its pathogenicity to that of the ancestral H6N1 virus A/Quail/HK/421/2002(H6N1). The Egyptian H6N1 virus replicated efficiently in C57BL/6 mice without prior adaptation and grew faster and reached higher titers than in A549 cells than the ancestral strain. These results show that reassortant H6 AIVs might pose a potential threat to human health and highlight the need to continue surveillance of H6 AIVs circulating in nature.

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.

Incidence and Seroprevalence of Avian Influenza in a Cohort of Backyard Poultry Growers, Egypt, August 2015-March 2019.

Currently enzootic avian influenza H5N1, H9N2, and H5N8 viruses were introduced into poultry in Egypt in 2006, 2011, and 2016, respectively. Infections with H5N1 and H9N2 were reported among poultry-exposed humans. We followed 2,402 persons from households raising backyard poultry from 5 villages in Egypt during August 2015-March 2019. We collected demographic, exposure, and health condition data and annual serum samples from each participant and obtained swab samples from participants reporting influenza-like illness symptoms. We performed serologic and molecular analyses and detected 4 cases of infection with H5N1 and 3 cases with H9N2. We detected very low seroprevalence of H5N1 antibodies and no H5N8 antibodies among the cohort; up to 11% had H9 antibodies. None of the exposure, health status, or demographic variables were related to being seropositive. Our findings indicate that avian influenza remains a public health risk in Eqypt, but infections may go undetected because of their mild or asymptomatic nature.

Evidence of infection with avian, human, and swine influenza viruses in pigs in Cairo, Egypt.

The majority of the Egyptian swine population was culled in the aftermath of the 2009 H1N1 pandemic, but small-scale growing remains. We sampled pigs from piggeries and an abattoir in Cairo. We found virological evidence of infection with avian H9N2 and H5N1 viruses as well as human pandemic H1N1 influenza virus. Serological evidence suggested previous exposure to avian H5N1 and H9N2, human pandemic H1N1, and swine avian-like and human-like viruses. This raises concern about potential reassortment of influenza viruses in pigs and highlights the need for better control and prevention of influenza virus infection in pigs.

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.

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.

Avian influenza A(H5N1) and A(H9N2) seroprevalence and risk factors for infection among Egyptians: a prospective, controlled seroepidemiological study.

BACKGROUND: A(H5N1) and A(H9N2) avian influenza viruses are enzootic in Egyptian poultry, and most A(H5N1) human cases since 2009 have occurred in Egypt. Our understanding of the epidemiology of avian viruses in humans remains limited. Questions about the frequency of infection, the proportion of infections that are mild or subclinical, and the case-fatality rate remain largely unanswered. METHODS: We conducted a 3-year, prospective, controlled, seroepidemiological study that enrolled 750 poultry-exposed and 250 unexposed individuals in Egypt. RESULTS: At baseline, the seroprevalence of anti-A(H5N1) antibodies (titer, ≥80) among exposed individuals was 2% significantly higher than that among the controls (0%). Having chronic lung disease was a significant risk factor for infection. Antibodies against A(H9N2) were not detected at baseline when A(H9N2) was not circulating in poultry. At follow-up, A(H9N2) was detected in poultry, and consequently, the seroprevalence among exposed humans was between 5.6% and 7.5%. Vaccination of poultry, older age, and exposure to ducks were risk factors for A(H9N2) infection. CONCLUSIONS: Results of this study indicate that the number of humans infected with avian influenza viruses is much larger than the number of reported confirmed cases. In an area where these viruses are enzootic in the poultry, human exposure to and infection with avian influenza becomes more common.

Equine influenza A(H3N8) virus isolated from Bactrian camel, Mongolia.

Because little is known about the ecology of influenza viruses in camels, 460 nasal swab specimens were collected from healthy (no overt illness) Bactrian camels in Mongolia during 2012. One specimen was positive for influenza A virus (A/camel/Mongolia/335/2012[H3N8]), which is phylogenetically related to equine influenza A(H3N8) viruses and probably represents natural horse-to-camel transmission.

Active surveillance for avian influenza virus, Egypt, 2010-2012.

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.

Incidence and neutralizing antibody seroprevalence of influenza B virus in Egypt: Results of a community-based cohort study.

Since 2000, two lineages of influenza B viruses, Victoria and Yamagata, have been circulating at similar frequencies worldwide. Little is known about the circulation of those viruses in Egypt. This study aims to describe the epidemiology of influenza B virus infections in Egypt, 2017-2019. This was performed through a household prospective cohort study on influenza infections among 2400 individuals from five villages. When a study participant had influenza like symptoms, a nasal swab and an oropharyngeal swab were obtained and tested by RT-PCR for influenza B infections. A serum sample was obtained from all participants annually to detect neutralizing antibodies using microneutralization assay. 9.1% of subjects were positive for influenza B viruses during season 2017-2018 mostly among preschoolers and 7.6% were positive during the season 2018-2019 with higher risk in females, potentially due to mothers being infected after contact with their children. The overall seroprevalence among the participants was 53.2% and 52.2% against the Victoria and Yamagata lineages respectively, the majority of seropositive participants were students. Multivariate analysis showed that age and having chronic diseases were the strongest predictors of infection. Our results show that both influenza B lineages circulated between 2017 and 2020 in Egypt almost in equal proportion. Encouraging the uptake of seasonal influenza vaccines is recommended.

Genetic and Antigenic Characteristics of Highly Pathogenic Avian Influenza A(H5N8) Viruses Circulating in Domestic Poultry in Egypt, 2017-2021.

In Egypt, the endemicity of avian influenza viruses is a serious concern. Since 2016, several outbreaks of H5N8 have been recorded among domestic poultry in various areas of the country. Active surveillance of domestic poultry across several governorates in Egypt from 2017 to 2021 detected at least six genotypes of Highly Pathogenic Avian Influenza (HPAI) H5N8 viruses with evidence of partial or complete annual replacement of dominant strains. Although all Egyptian H5N8 viruses had clade 2.3.4.4b hemagglutinin (HA) genes, the remaining viral gene segments were from multiple geographic origins, indicating that the H5N8 isolates resulted from multiple introductions. Mutations in the viral proteins associated with pathogenicity and antiviral drug resistance were detected. Some mutations in the HA resulted in antigenic drift. Heterogeneity in circulating H5N8 HPAI threatens poultry production and public health.

Insights into Genetic Characteristics and Virological Features of Endemic Avian Influenza A (H9N2) Viruses in Egypt from 2017-2021.

From 2010 to 2013, genotype I avian influenza A(H9N2) viruses of the G1-lineage were isolated from several poultry species in Egypt. In 2014, novel reassortant H9N2 viruses were detected in pigeons designated as genotype II. To monitor the subsequent genetic evolution of Egyptian A(H9N2) viruses, we characterized the full genomes of 173 viruses isolated through active surveillance from 2017 to 2022. In addition, we compared the virological characteristics and pathogenicity of representative viruses. Phylogenetic analysis of the HA indicated that all studied sequences from 2017-2021 were grouped into G1-like H9N2 viruses previously detected in Egypt. Phylogenetic analysis indicated that the Egyptian A(H9N2) viruses had undergone further reassortment, inheriting four genes (PB2, PB1, PA, NS) from genotype II, with their remaining segments deriving from genotype I viruses (these viruses designated as genotype III). Studying the virological features of the two most dominant genotypes (I and III) of Egyptian H9N2 viruses in vitro and in vivo indicated that both replicated well in mammalian cells, but did not show any clinical signs in chickens, ducks, and mice. Monitoring avian influenza viruses through surveillance programs and understanding the genetic and antigenic characteristics of circulating H9N2 viruses are essential for risk assessment and influenza pandemic preparedness.

Highly Pathogenic Avian Influenza A(H5N1) Virus Clade 2.3.4.4b in Wild Birds and Live Bird Markets, Egypt.

Clade 2.3.4.4 H5Nx influenza viruses have further diversified into several subclades. Sub-clade 2.3.4.4b H5N1 viruses have been widely circulating in wild birds and detected in Europe, Africa, Asia, and North America since October 2020. In this study, we report the first detection of highly pathogenic avian influenza H5N1 clade 2.3.4.4b viruses in wild birds and domestic ducks from live bird markets in Egypt. Phylogenetic analysis revealed that the Egyptian H5N1 virus retained the genomic composition of Eurasian strains. Mutations in the viral proteins associated with zoonotic potential and pathogenicity were detected in Egyptian isolates. Egypt is considered a hot spot for the evolution of the influenza virus, so active surveillance of avian influenza viruses in Egypt is warranted.

Continuing threat of influenza (H5N1) virus circulation in Egypt.

Reservoirs for the continuing influenza (H5N1) outbreaks in Egypt are ill-defined. Through active surveillance, we detected highly pathogenic influenza subtype H5 viruses in all poultry sectors; incidence was 5%. No other subtypes were found. Continued circulation of influenza (H5N1) viruses in various regions and poultry sectors perpetuates human exposure in Egypt.

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.

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 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.

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.