Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses.

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.

Preferential Selection and Contribution of Non-Structural Protein 1 (NS1) to the Efficient Transmission of Panzootic Avian Influenza H5N8 Virus Clades 2.3.4.4A and B in Chickens and Ducks.

Highly pathogenic avian influenza virus H5N8 clade 2.3.4.4 caused outbreaks in poultry at an unprecedented global scale. The virus was spread by wild birds in Asia in two waves: clade 2.3.4.4A in 2014/2015 and clade 2.3.4.4B from 2016 up to today. Both clades were highly virulent in chickens, but only clade B viruses exhibited high virulence in ducks. Viral factors which contribute to virulence and transmission of these panzootic H5N8 2.3.4.4 viruses are largely unknown. The NS1 protein, typically composed of 230 amino acids (aa), is a multifunctional protein which is also a pathogenicity factor. Here, we studied the evolutionary trajectory of H5N8 NS1 proteins from 2013 to 2019 and their role in the fitness of H5N8 viruses in chickens and ducks. Sequence analysis and in vitro experiments indicated that clade 2.3.4.4A and clade 2.3.4.4B viruses have a preference for NS1 of 237 aa and 217 aa, respectively, over NS1 of 230 aa. NS217 was exclusively seen in domestic and wild birds in Europe. The extension of the NS1 C terminus (CTE) of clade B virus reduced virus transmission and replication in chickens and ducks and partially impaired the systemic tropism to the endothelium in ducks. Conversely, lower impact on fitness of clade A virus was observed. Remarkably, the NS1 of clade A and clade B, regardless of length, was efficient in blocking interferon (IFN) induction in infected chickens, and changes in the NS1 C terminus reduced the efficiency for interferon antagonism. Together, the NS1 C terminus contributes to the efficient transmission and high fitness of H5N8 viruses in chickens and ducks. IMPORTANCE The panzootic H5N8 highly pathogenic avian influenza viruses of clade 2.3.4.4A and 2.3.4.4B devastated the poultry industry globally. Clade 2.3.4.4A was predominant in 2014/2015 while clade 2.3.4.4B was widely spread in 2016/2017. The two clades exhibited different pathotypes in ducks. Virus factors contributing to virulence and transmission are largely unknown. The NS1 protein is typically composed of 230 amino acids (aa) and is an essential interferon (IFN) antagonist. Here, we found that the NS1 protein of clade 2.3.4.4A preferentially evolved toward long NS1 with 237 aa, while clade 2.3.4.4B evolved toward shorter NS1 with 217 aa (exclusively found in Europe) due to stop codons in the C terminus (CTE). We showed that the NS1 CTE of H5N8 is required for efficient virus replication, transmission, and endotheliotropism in ducks. In chickens, H5N8 NS1 evolved toward higher efficiency to block IFN response. These findings may explain the preferential pattern for short NS1 and high fitness of the panzootic H5N8 in birds.

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.

Biological characterization of highly pathogenic avian influenza H5N1 viruses that infected humans in Egypt in 2014-2015.

Highly pathogenic avian influenza (HPAI) H5N1 influenza viruses emerged as a human pathogen in 1997 with expected potential to undergo sustained human-to-human transmission and pandemic viral spread. HPAI H5N1 is endemic in Egyptian poultry and has caused sporadic human infection. The first outbreak in early 2006 was caused by clade 2.2 viruses that rapidly evolved genetically and antigenically. A sharp increase in the number of human cases was reported in Egypt in the 2014/2015 season. In this study, we analyzed and characterized three isolates of HPAI H5N1 viruses isolated from infected humans in Egypt in 2014/2015. Phylogenetic analysis demonstrated that the nucleotide sequences of eight segments of the three isolates were clustered with those of members of clade 2.2.1.2. We also found that the human isolates from 2014/2015 had a slight, non-significant difference in their affinity for human-like sialic acid receptors. In contrast, they showed significant differences in their replication kinetics in MDCK, MDCK-SIAT, and A549 cells as well as in embryonated chicken eggs. An antiviral bioassay study revealed that all of the isolates were susceptible to amantadine. Therefore, further investigation and monitoring is required to correlate the genetic and/or antigenic changes of the emerging HPAI H5N1 viruses with possible alteration in their characteristics and their potential to become a further threat to public health.

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.

Molecular characterization of avian influenza H5N1 virus in Egypt and the emergence of a novel endemic subclade.

Clade 2.2 highly pathogenic H5N1 viruses have been in continuous circulation in Egyptian poultry since 2006. Their persistence caused significant genetic drift that led to the reclassification of these viruses into subclades 2.2.1 and 2.2.1.1. Here, we conducted full-genome sequence and phylogenetic analyses of 45 H5N1 isolated during 2006-2013 through systematic surveillance in Egypt, and 53 viruses that were sequenced previously and available in the public domain. Results indicated that H5N1 viruses in Egypt continue to evolve and a new distinct cluster has emerged. Mutations affecting viral virulence, pathogenicity, transmission, receptor-binding preference and drug resistance were studied. In light of our findings that H5N1 in Egypt continues to evolve, surveillance and molecular studies need to be sustained.

Genetic and antigenic evolution of H9N2 avian influenza viruses circulating in Egypt between 2011 and 2013.

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.

Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs.

IMPORTANCE: We present the first study of the 3D kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the early host response in a large lung volume using a combination of tissue imaging and transcriptomics. This approach allowed us to make a number of important findings: Spatially restricted antiviral response is shown, including the formation of monocytic macrophage clusters and upregulation of the major histocompatibility complex II in infected epithelial cells. The monocyte-derived macrophages are linked to SARS-CoV-2 clearance, and the appearance of these cells is associated with post-infection endothelial damage; thus, we shed light on the role of these cells in infected tissue. An early onset of tissue repair occurring simultaneously with inflammatory and necrotizing processes provides the basis for longer-term alterations in the lungs.

Characterization of the recent outbreak of foot-and-mouth disease virus serotype SAT2 in Egypt.

An outbreak of foot-and-mouth disease (FMD) in Egypt affected approximately 40,000 cattle and water buffaloes and killed more than 4,600 animals during February-March 2012. To investigate the etiology of the 2012 outbreak, specimens were collected from six governorates and analyzed using universal primers to amplify the 5' untranslated region (UTR) by reverse-transcription polymerase chain reaction. Only FMDV-SAT2 was detected, with an overall detection rate of 80.3 %. Complete VP1- and leader-proteinase-coding sequences, obtained from three isolates from three different governorates, were compared with previously reported sequences. Phylogenetic analysis of these sequences indicated that the circulating viruses were homogeneous and were closely related to topotype VII. Importantly, the newly emerged viruses were genetically closely related to strains isolated from Saudi Arabia, Sudan, Eritrea and Cameroon between 2000 and 2010, suggesting the dominant nature of this virus and underscoring the need for worldwide intensive surveillance to minimize its devastating consequences.

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.

Prevalence of viral pathogens in a sample of hospitalized Egyptian children with acute lower respiratory tract infections: a two-year prospective study.

Background: Viral pneumonias are a major cause of childhood mortality. Proper management needs early and accurate diagnosis. This study objective is to investigate the viral etiologies of pneumonia in children. Results: This prospective study enrolled 158 and 101 patients in the first and second year, respectively, and their mean age was 4.72 ± 2.89. Nasopharyngeal swabs were collected and subjected to virus diagnosis by reverse transcription polymerase chain reaction (RT-PCR). Viral etiologies of pneumonia were evidenced in 59.5% of the samples in the first year, all of them were affirmative for influenza A, 2 samples were affirmative for Human coronavirus NL63, and one for Human coronavirus HKU1. In the second year, 87% of patients had a viral illness. The most prevalent agents are human metapneumovirus which was detected in 44 patients (43.6%) followed by human rhinovirus in 35 patients (34.7%) and then parainfluenza-3 viruses in 33 patients (32.7%), while 14 patients had a confirmed diagnosis for both Pan coronavirus and Flu-B virus. Conclusions: Viral infection is prevalent in the childhood period; however, the real magnitude of viral pneumonia in children is underestimated. The reverse transcriptase polymerase chain reaction has to be a vital tool for epidemiological research and is able to clear the gaps in-between clinical pictures and final diagnoses.

The role of glycosylation in the N-terminus of the hemagglutinin of a unique H4N2 with a natural polybasic cleavage site in virus fitness in vitro and in vivo.

To date, only low pathogenic (LP) H5 and H7 avian influenza viruses (AIV) have been observed to naturally shift to a highly pathogenic (HP) phenotype after mutation of the monobasic hemagglutinin (HA) cleavage site (HACS) to polybasic motifs. The LPAIV monobasic HACS is activated by tissue-restricted trypsin-like enzymes, while the HPAIV polybasic HACS is activated by ubiquitous furin-like enzymes. However, glycosylation near the HACS can affect proteolytic activation and reduced virulence of some HPAIV in chickens. In 2012, a unique H4N2 virus with a polybasic HACS was isolated from quails but was LP in chickens. Whether glycosylation sites (GS) near the HACS hinder the evolution of HPAIV H4N2 remains unclear. Here, we analyzed the prevalence of potential GS in the N-terminus of HA1, 2NYT4 and 18NGT20, in all AIV sequences and studied their impact on H4N2 virus fitness. Although the two motifs are conserved, some non-H5/H7 subtypes lack one or both GS. Both sites were glycosylated in this H4N2 virus. Deglycosylation increased trypsin-independent replication in cell culture, cell-to-cell spread and syncytium formation at low-acidic pH, but negatively affected the thermostability and receptor-binding affinity. Alteration of 2NYT4 with or without 18NGT20 enabled systemic spread of the virus to different organs including the brain of chicken embryos. However, all intranasally inoculated chickens did not show clinical signs. Together, although the conserved GS near the HACS are important for HA stability and receptor binding, deglycosylation increased the H4N2 HA-activation, replication and tissue tropism suggesting a potential role for virus adaptation in poultry.

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.

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.

Re-emergence of amantadine-resistant variants among highly pathogenic avian influenza H5N1 viruses in Egypt.

Highly pathogenic avian influenza (HPAI) H5N1 virus continues to undergo substantial evolution. Emergence of antiviral resistance among H5N1 avian influenza viruses is a major challenge in the control of pandemic influenza. Numerous studies have focused on the genetic and evolutionary dynamics of the hemagglutinin and neuraminidase genes; however, studies on the susceptibility of HPAI H5N1 viruses to amantadine and genetic diversity of the matrix (M) gene are limited. Accordingly, we studied the amantadine susceptibility of the HPAI H5N1 viruses isolated in Egypt during 2006-2015 based on genotypic and phenotypic characteristics. We analyzed data on 253 virus sequences and constructed a phylogenetic tree to calculate selective pressures on sites in the M2 gene associated with amantadine-resistance among different clades. Selection pressure was identified in the transmembrane domain of M2 gene at positions 27 and 31. Amantadine-resistant variants emerged in 2007 but were not circulating between 2012 and 2014. By 2015, amantadine-resistant HPAI H5N1 viruses re-emerged. This may be associated with the uncontrolled prescription of amantadine for prophylaxis and control of avian influenza infections in the poultry farm sector in Egypt. More epidemiological research is required to verify this observation.

Proteolytic enzymes in embryonated chicken eggs sustain the replication of egg-grown low-pathogenicity avian influenza viruses in cells in the absence of exogenous proteases.

Low pathogenic influenza viruses grow readily in embryonated chicken eggs but require the addition of exogenous proteases to grow in MDCK cell culture. In this study, we found that the influenza viruses propagated previously in eggs, can grow for up to two passages in cell culture without the addition of exogenous proteolytic enzymes. These results indicate that the reason for virus propagation in cells during the first two passages may be due to proteases from egg allantoic fluid carried over from egg culture. The ability of influenza viruses to grow in cells in the absence of trypsin is currently considered as a hallmark of highly pathogenic influenza viruses. Our data indicate that differentiating between high and low pathogenicity using cell culture only is not appropriate and other indicators such as sequence analysis and in vitro pathogenicity index should be performed.