Highly Pathogenic Avian Influenza Virus (H5N8) Clade 2.3.4.4 Infection in Migratory Birds, Egypt.

We isolated highly pathogenic avian influenza virus (H5N8) of clade 2.3.4.4 from the common coot (Fulica atra) in Egypt, documenting its introduction into Africa through migratory birds. This virus has a close genetic relationship with subtype H5N8 viruses circulating in Europe. Enhanced surveillance to detect newly emerging viruses is warranted.

Heterologous post-infection immunity against Egyptian avian influenza virus (AIV) H9N2 modulates the course of subsequent infection by highly pathogenic AIV H5N1, but vaccination immunity does not.

In Egypt, zoonotic A/goose/Guangdong/1/96 (gs/GD-like) highly pathogenic avian influenza virus (HPAIV) H5N1 of clade 2.2.1.2 is entrenched in poultry populations and has co-circulated with low-pathogenic avian influenza virus H9N2 of the G1 lineage since 2010. Here, the impact of H9N2 infection or vaccination on the course of consecutive infection with a lethal Egyptian HPAIV H5N1 is studied. Three-week-old chickens were infected with H9N2 or vaccinated with inactivated H9N2 or H5N1 antigens and challenged three weeks later by an HPAIV H5N1. Interestingly, pre-infection of chickens with H9N2 decreased the oral excretion of H5N1 to levels that were comparable to those of H5N1-immunized chickens, but vaccination with inactivated H9N2 did not. H9N2 pre-infection modulated but did not conceal clinical disease by HPAIV H5N1. By contrast, homologous H5 vaccination abolished clinical syndromic surveillance, although vaccinated clinical healthy birds were capable of spreading the virus.

A genetically engineered H5 protein expressed in insect cells confers protection against different clades of H5N1 highly pathogenic avian influenza viruses in chickens.

The evolution of highly pathogenic H5N1 avian influenza viruses (HPAI-H5N1) has resulted in the appearance of a number of diverse groups of HPAI-H5N1 based on the presence of genetically similar clusters of their haemagglutinin sequences (clades). An H5 antigen encoded by a recombinant baculovirus and expressed in insect cells was used for oil-emulsion-based vaccine prototypes. In several experiments, vaccination was performed at 10 days of age, followed by challenge infection on day 21 post vaccination (PV) with HPAI-H5N1 clades 2.2, 2.2.1, and 2.3.2. A further challenge infection with HPAI-H5N1 clade 2.2.1 was performed at day 42 PV. High haemagglutination inhibition titres were observed for the recH5 vaccine antigen, and lower haemagglutination inhibition titres for the challenge virus antigens. Nevertheless, the rate of protection from mortality and clinical signs was 100% when challenged at 21 days PV and 42 days PV, indicating protection over the entire broiler chicken rearing period without a second vaccination. The unvaccinated control chickens mostly died between two and five days after challenge infection. A low level of viral RNA was detected by reverse transcription followed by a quantitative polymerase chain reaction in a limited number of birds for a short period after challenge infection, indicating a limited spread of HPAI-H5N1 at flock level. Furthermore, it was observed that the vaccine can be used in a differentiation infected from vaccinated animals (DIVA) approach, based on the detection of nucleoprotein antibodies in vaccinated/challenged chickens. The vaccine fulfilled all expectations of an inactivated vaccine after one vaccination against challenge with different clades of H5N1-HPAI and is suitable for a DIVA approach.

Phylodynamics of avian influenza clade 2.2.1 H5N1 viruses in Egypt.

BACKGROUND: Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are widely distributed within poultry populations in Egypt and have caused multiple human infections. Linking the epidemiological and sequence data is important to understand the transmission, persistence and evolution of the virus. This work describes the phylogenetic dynamics of H5N1 based on molecular characterization of the hemagglutinin (HA) gene of isolates collected from February 2006 to May 2014. METHODS: Full-length HA sequences of 368 H5N1 viruses were generated and were genetically analysed to study their genetic evolution. They were collected from different poultry species, production sectors, and geographic locations in Egypt. The Bayesian Markov Chain Monte Carlo (BMCMC) method was applied to estimate the evolutionary rates among different virus clusters; additionally, an analysis of selection pressures in the HA gene was performed using the Single Likelihood Ancestor Counting (SLAC) method. RESULTS: The phylogenetic analysis of the H5 gene from 2006-14 indicated the presence of one virus introduction of the classic clade (2.2.1) from which two main subgroups were originated, the variant subgroup which was further subdivided into 2 sub-divisions (2.2.1.1 and 2.2.1.1a) and the endemic subgroup (2.2.1.2). The clade 2.2.1.2 showed a high evolution rate over a period of 6 years (6.9 × 10(-3) sub/site/year) in comparison to the 2.2.1.1a variant cluster (7.2 × 10(-3) over a period of 4 years). Those two clusters are under positive selection as they possess 5 distinct positively selected sites in the HA gene. The mutations at 120, 154, and 162 HA antigenic sites and the other two mutations (129∆, I151T) that occurred from 2009-14 were found to be stable in the 2.2.1.2 clade. Additionally, 13 groups of H5N1 HPAI viruses were identified based on their amino acid sequences at the cleavage site and "EKRRKKR" became the dominant pattern beginning in 2013. CONCLUSIONS: Continuous evolution of H5N1 HPAI viruses in Egypt has been observed in all poultry farming and production systems in almost all regions of the country. The wide circulation of the 2.2.1.2 clade carrying triple mutations (120, 129∆, I151T) associated with increased binding affinity to human receptors is an alarming finding of public health importance.

The sequence of the full spike S1 glycoprotein of infectious bronchitis virus circulating in Egypt reveals evidence of intra-genotypic recombination.

Infectious bronchitis virus (IBV) continues to circulate worldwide, with a significant impact on the poultry industry and affecting both vaccinated and unvaccinated flocks. Several studies have focused on the hypervariable regions (HVRs) of the spike gene (S1); however, genetic and bioinformatics studies of the whole S1 gene are limited. In this study, the whole S1 gene of five Egyptian IBVs was genetically analyzed. Phylogenetic analysis revealed that the Egyptian IBVs are clustered within two distinct groups: the classic group resembling the GI-1 genotype (vaccine strains) and the variant group (field strains) of the GI-23 genotype. The variant genotype was divided into two distinct subgroups (Egy/var I and Egy/var II) resembling the Israeli variants IS/1494 and IS885 strain, respectively. Significant amino acid sequence differences between the two subgroups, especially in the epitope sites, were identified. A deletion at position 63 and an I69A/S substitution mutation associated with virus tropism were detected in the receptor-binding sites. The deduced amino acid sequence of HVRs of the variant subgroups indicated different genetic features in comparison to the classic vaccine group (H120 lineage). The Egyptian variant IBVs also contained additional N-glycosylation sites compared to the classical viruses. Recombination analysis gave evidence for distinct patterns of origin by recombination throughout the S1 gene, suggesting that the recent virus IBV-EG/1586CV-2015 emerged as a recombinant of two viruses from the variant groups Egy/var I and Egy/var II, providing another example of intra-genotypic recombination among IBVs and the first example of recombination within the GI-23 genotype. Our data suggest that both mutation and recombination may be contributing to the emergence of IBV variants. Moreover, we found that the commercially used vaccines are genotypically distant from the circulating field strains. Hence, continuous follow-up of the current vaccine strategy is highly recommended for better control and prevention of infectious bronchitis virus in the poultry sector in Egypt.

The matrix gene of influenza A H5N1 in Egypt, 2006-2016: molecular insights and distribution of amantadine-resistant variants.

Large-scale sequence analysis of Matrix (M) gene and its coding proteins M1 and M2 was performed for 274 highly pathogenic avian influenza viruses H5N1 circulated in Egypt from 2006 to 2016. The aim is to study the amantadine-resistant markers distribution and to estimate the evolutionary rate. 246 viruses were obtained from the Global Initiative on Sharing All Influenza Data base, and 28 additional viruses were sequenced. Maximum clade credibility (MCC) phylogenetic tree revealed that the M gene has evolved into two different lineages. Estimated Evolutionary analysis showed that the M2 protein possessed higher evolutionary rates (3.45 × 10-3) than the M1 protein (2.73 × 10-3). M gene encoding proteins revealed significant markers described to be associated with host tropism and increase in virulence: V15I, N30D, and T121A in M1 and L55F in M2 protein. Site analysis focusing attention on the temporal and host distribution of the amantadine-resistant markers was carried out and showed that vast majority of the M2 amantadine-resistant variants of clade 2.2.1.1 (n = 90) is N31 marker, in addition to G27 (n = 7), A27 (n = 5), I27 (n = 1), and S30 (n = 1). In 2010-2011, amantadine resistant frequency increased considerably resembling more than half of the resistant variants. Notably, all viruses of clade 2.2.1.1 possessed amantadine-resistant marker. However, almost all current circulating viruses in Egypt of clade 2.2.1.2 from 2014 to 2016 did not carry any amantadine-resistant markers.

Surveillance on A/H5N1 virus in domestic poultry and wild birds in Egypt.

BACKGROUND: The endemic H5N1 high pathogenicity avian influenza virus (A/H5N1) in poultry in Egypt continues to cause heavy losses in poultry and poses a significant threat to human health. METHODS: Here we describe results of A/H5N1 surveillance in domestic poultry in 2009 and wild birds in 2009-2010. Tracheal and cloacal swabs were collected from domestic poultry from 22024 commercial farms, 1435 backyards and 944 live bird markets (LBMs) as well as from 1297 wild birds representing 28 different types of migratory birds. Viral RNA was extracted from a mix of tracheal and cloacal swabs media. Matrix gene of avian influenza type A virus was detected using specific real-time reverse-transcription polymerase chain reaction (RT-qPCR) and positive samples were tested by RT-qPCR for simultaneous detection of the H5 and N1 genes. RESULTS: In this surveillance, A/H5N1 was detected from 0.1% (n = 23/) of examined commercial poultry farms, 10.5% (n = 151) of backyard birds and 11.4% (n = 108) of LBMs but no wild bird tested positive for A/H5N1. The virus was detected from domestic poultry year-round with higher incidence in the warmer months of summer and spring particularly in backyard birds. Outbreaks were recorded mostly in Lower Egypt where 95.7% (n = 22), 68.9% (n = 104) and 52.8% (n = 57) of positive commercial farms, backyards and LBMs were detected, respectively. Higher prevalence (56%, n = 85) was reported in backyards that had mixed chickens and waterfowl together in the same vicinity and LBMs that had waterfowl (76%, n = 82). CONCLUSION: Our findings indicated broad circulation of the endemic A/H5N1 among poultry in 2009 in Egypt. In addition, the epidemiology of A/H5N1 has changed over time with outbreaks occurring in the warmer months of the year. Backyard waterfowl may play a role as a reservoir and/or source of A/H5N1 particularly in LBMs. The virus has been established in poultry in the Nile Delta where major metropolitan areas, dense human population and poultry stocks are concentrated. Continuous surveillance, tracing the source of live birds in the markets and integration of multifaceted strategies and global collaboration are needed to control the spread of the virus in Egypt.

Emergence of Highly Pathogenic Avian Influenza A Virus (H5N1) of Clade 2.3.4.4b in Egypt, 2021-2022.

Wild migratory birds have the capability to spread avian influenza virus (AIV) over long distances as well as transmit the virus to domestic birds. In this study, swab and tissue samples were obtained from 190 migratory birds during close surveillance in Egypt in response to the recent outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus. The collected samples were tested for a variety of AIV subtypes (H5N1, H9N2, H5N8, and H6N2) as well as other pathogens such as NDV, IBV, ILT, IBDV, and WNV. Among all of the tested samples, the HPAI H5N1 virus was found in six samples; the other samples were found to be negative for all of the tested pathogens. The Egyptian HPAI H5N1 strains shared genetic traits with the HPAI H5N1 strains that are currently being reported in Europe, North America, Asia, and Africa in 2021-2022. Whole genome sequencing revealed markers associated with mammalian adaption and virulence traits among different gene segments, similar to those found in HPAI H5N1 strains detected in Europe and Africa. The detection of the HPAI H5N1 strain of clade 2.3.4.4b in wild birds in Egypt underlines the risk of the introduction of this strain into the local poultry population. Hence, there is reason to be vigilant and continue epidemiological and molecular monitoring of the AIV in close proximity to the domestic-wild bird interface.

Isolation of avian influenza H5N1 virus from vaccinated commercial layer flock in Egypt.

BACKGROUND: Uninterrupted transmission of highly pathogenic avian influenza virus (HPAIV) H5N1 of clade 2.2.1 in Egypt since 2006 resulted in establishment of two main genetic clusters. The 2.2.1/C group where all recent human and majority of backyard origin viruses clustered together, meanwhile the majority of viruses derived from vaccinated poultry in commercial farms grouped in 2.2.1.1 clade. FINDINGS: In the present investigation, an HPAIV H5N1 was isolated from twenty weeks old layers chickens that were vaccinated with a homologous H5N1 vaccine at 1, 7 and 16 weeks old. At twenty weeks of age, birds showed cyanosis of comb and wattle, decrease in egg production and up to 27% mortality. Examined serum samples showed low antibody titer in HI test (Log2 3.2 ± 4.2). The hemagglutinin (HA) and neuraminidase (NA) genes of the isolated virus were closely related to viruses in 2.2.1/C group isolated from poultry in live bird market (LBM) and backyards or from infected people. Conspicuous mutations in the HA and NA genes including a deletion within the receptor binding domain in the HA globular head region were observed. CONCLUSIONS: Despite repeated vaccination of layer chickens using a homologous H5N1 vaccine, infection with HPAIV H5N1 resulted in significant morbidity and mortality. In endemic countries like Egypt, rigorous control measures including enforcement of biosecurity, culling of infected birds and constant update of vaccine virus strains are highly required to prevent circulation of HPAIV H5N1 between backyard birds, commercial poultry, LBM and humans.

Isolation of H9N2 avian influenza virus from bobwhite quail (Colinus virginianus) in Egypt.

This study describes the first isolation of H9N2 avian influenza virus (AIV) from commercial bobwhite quail (Colinus virginianus) in Egypt. Infected birds showed neither clinical signs nor mortality. Virus isolation and real-time reverse transcription polymerase chain reaction confirmed the presence of the H9N2 virus in cloacal swab samples collected at 35 days of age and the absence of other AIV subtypes, including H5 and H7. The hemagglutinin and neuraminidase genes of the isolated virus showed 99.1% and 98.2% nucleotide identity and 97.3% and 100% amino acid identity, respectively, to those of H9N2 viruses currently circulating in poultry in the Middle East. Phylogenetically, the Egyptian H9N2 virus was closely related to viruses of the G1-like lineage isolated from neighbouring countries, indicating possible epidemiological links.

Distribution of avian influenza H5N1 viral RNA in tissues of AI-vaccinated and unvaccinated contact chickens after experimental infection.

Avian influenza due to highly pathogenic avian influenza (HPAIV) H5N1 virus is not a food-borne illness but a serious panzootic disease with the potential to be pandemic. In this study, broiler chickens were vaccinated with commercial H5N1 or H5N2 inactivated vaccines prior to being challenged with an HPAIV H5N1 (clade 2.2.1 classic) virus. Challenged and non-challenged vaccinated chickens were kept together, and unvaccinated chickens served as contact groups. Post-challenge samples from skin and edible internal organs were collected from dead and sacrificed (after a 14-day observation period) birds and tested using qRT-PCR for virus detection and quantification. H5N1 vaccine protected chickens against morbidity, mortality and transmission. Virus RNA was not detected in the meat or edible organs of chickens vaccinated with H5N1 vaccine. Conversely, H5N2 vaccine did not confer clinical protection, and a significant virus load was detected in the meat and internal organs. Phylogenetic analysis showed that the H5N1 virus vaccine and challenge virus strains are closely related. The results of the present study strongly suggest a need for proper selection of vaccines and their routine evaluation against newly emergent field viruses. These actions will help to reduce human exposure to HPAIV H5N1 virus from both infected live birds and slaughtered poultry. In addition, rigorous preventive measures should be put in place in order to minimize the public-health risks of avian influenza at the human-animal interface.

Complete genome characterization of avian influenza virus subtype H9N2 from a commercial quail flock in Egypt.

The suspected presence of avian influenza virus subtype H9N2 in poultry in Egypt is a major concern since this subtype is widely distributed in different countries in the Middle East, here we describe the full genetic characterization of an avian influenza A virus (Qa/Egypt/11; H9N2) of subtype H9N2 that was previously isolated from a clinically normal quail flock in Giza, Egypt in May 2011. The nucleotide sequence analysis of the hemagglutinin gene of the isolated Egyptian virus showed the highest similarity with one group of recent Israeli strains (97 %) circulating from 2006-2010. Sequence homology and phylogenetic analysis indicated that the Qa/Egypt/11 isolate belonged to the A/quail/Hong Kong/G1/1997-like lineage with new mutations identified in all viral proteins. The phylogenetic analysis for the eight genes indicated placement of the Egyptian virus within the same lineage of H9N2 viruses that circulated in the region from 2006, especially with one group of recent Israeli strains. However, phylogenetic analysis of the internal genes like PB2, NP, and PA genes identified possible reassortment events for these genes with singular Israeli strains. This study indicates progressive evolution of this subtype in the Middle East region and possible mechanism of virus adaptation in land-based poultry like in quails.

Diversifying evolution of highly pathogenic H5N1 avian influenza virus in Egypt from 2006 to 2011.

An evolutionary analysis was conducted of 354 hemagglutinin (HA) and 208 neuraminidase (NA) genes, including newly generated sequences of 5 HA and 30 NA, of Egyptian H5N1 clade 2.2.1 viruses isolated from poultry and humans. Five distinct phylogenetically distinguishable clusters arose from a monophyletic origin since 2006. Only two clusters remained in circulation after 2009: (i) A cluster of viruses arose in 2007 in industrial-vaccinated chickens and carried multiple mutations in or adjacent to the immunogenic epitopes of the HA. Viruses within this cluster evolved with significantly elevated mutation rates indicating persisting selective pressures, e.g. to escape host immunity and (ii) The second group arose in 2008 and harboured strains from recent human infections featuring a conspicuous deletion in the HA receptor-binding domain and substitutions close to the highly conserved active site of the NA. In both sublineages, a number of positively selected amino acids, different glycosylation patterns and variations in the polybasic proteolytic cleavage site were observed. Continuous monitoring of the evolving H5N1 virus in Egypt is essential to develop new control campaigns in poultry and human population.

Effect of cocirculation of highly pathogenic avian influenza H5N1 subtype with low pathogenic H9N2 subtype on the spread of infections.

Widespread prevalence of avian influenza H9N2 subtype in the Middle East region and its detection in Egypt in quail in early summer 2011 added another risk factor to the Egyptian poultry industry in addition to highly pathogenic H5N1 subtype. This situation increases the need for further surveillance and investigation of H9N2 viruses in commercial and household chickens. This work describes detection and genetic characterization of recently isolated H9N2 viruses from chicken flocks. Parallel detection and genetic characterization of H5N1 viruses from infections in poultry has also been done to compare the prevalence of the two subtypes in close geographic locations in Egypt. Phylogenetic analysis of the HA gene showed that the Egyptian isolates of H9N2 were grouped together within the quail/Hong Kong/G1/97-like lineage, similar to the circulating viruses in the Middle East, with very close phylogeny to the Israeli viruses. The prevalence of H5N1 viruses from cases recorded in poultry in the nearby areas revealed a marked decrease in disease incidence in commercial broilers but an increased incidence in household birds. The genetic characterization of the H5N1 viruses indicated predominance of the classic 2.2.1 subclade, with evolution of new viruses and no detection for the variant 2.2.1.1 subclade. The cocirculation of the two subtypes, H5N1 and H9N2, of avian influenza may affect the limit of spread and the epizootiologic pattern of the infections for both subtypes, especially when different vaccination and biosecurity approaches are applied in the field level.

Comparison of genomic and antigenic properties of Newcastle Disease virus genotypes II, XXI and VII from Egypt do not point to antigenic drift as selection marker.

Newcastle disease (ND), caused by avian orthoavulavirus type-1 (NDV), is endemic in poultry in many regions of the world and causes continuing outbreaks in poultry populations. In the Middle East, genotype XXI, used to be present in poultry in Egypt but has been replaced by genotype VII. We investigated whether virus evolution contributed to superseding and focussed on the antigenic sites within the hemagglutinin-neuraminidase (HN) spike protein. Full-length sequences of an NDV genotype VII isolate currently circulating in Egypt was compared to a genotype XXI isolate that was present as co-infection with vaccine-type viruses (II) in a historical virus isolated in 2011. Amino acid differences in the HN glycoprotein for both XXI and VII viruses amounted to 11.7% and 11.9%, respectively, compared to the La Sota vaccine type. However, mutations within the globular head (aa 126-570), bearing relevant antigenic sites, were underrepresented (a divergence of 8.8% and 8.1% compared to 22.4% and 25.6% within the protein domains encompassing cytoplasmic tail, transmembrane part and stalk regions (aa 1-125) for genotypes XXI and VII, respectively). Nevertheless, reaction patterns of HN-specific monoclonal antibodies inhibiting receptor binding revealed differences between vaccine-type viruses and genotype XXI and VII viruses for epitopes located in the head domain. Accordingly, compared to Egyptian vaccine-type isolates and the La Sota vaccine reference strain, single aa substitutions in 6 of 10 described neutralizing epitopes of HN were found. However, the same alterations in neutralization sensitive epitopes were present in old genotype XXI as well as in newly emerged genotype VII isolates. In addition, isolates were indistinguishable by polyclonal chicken sera raised against different genotypes including vaccine viruses. These findings suggest that factors other than antigenic differences within the HN protein account for facilitating the spread of genotype VII versus genotype XXI viruses in Egypt.

Molecular Epidemiology and Evolutionary Analysis of Avian Influenza A(H5) Viruses Circulating in Egypt, 2019-2021.

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019-2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019-2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020-2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt.

Novel reassortant of H9N2 avian influenza viruses isolated from chickens and quails in Egypt.

BACKGROUND AND AIM: Poultry infections with H9N2 avian influenza viruses (AIVs) are endemic in Egypt. This study determined the genetic changes in the sequences of H9N2 AIVs isolated from chicken and quails in Egypt, including determining genetic reassortment and detecting the main genetic changes in hemagglutinin (HA) and neuraminidase (NA) genes. MATERIALS AND METHODS: Swab samples were collected from chicken and quails, examined through reverse transcription-polymerase chain reaction, and AIVs from positive samples were isolated in embryonated chicken eggs. Complete genome sequencing and phylogenetic analyses were conducted for two H9N2 AIV isolates, and sequences of HA and NA gene segments were analyzed in another two isolates. RESULTS: A novel reassortant virus was identified from a commercial chicken flock (A/chicken/Egypt/374V/2016) and quails from a live bird market (A/quail/Egypt/1253V/2016). The reassortant viruses acquired four genome segments from the classic Egyptian H9N2 viruses (HA, NA, NP, and M) and four segments from Eurasian AIVs (PB2, PB1, PA, and NS). Many genetic changes have been demonstrated in HA and NA genes. The isolated novel reassortant H9N2 virus from quails showed amino acid mutations in the antigenic sites on the globular head of the mature HA monomer matched with the parent Egyptian H9N2 virus. CONCLUSION: This work described the genetic characterization of a novel reassortment of the H9N2 virus in Egypt. The emergence of new reassorted AIV viruses and genome variability raises the concern of an influenza pandemic with zoonotic potentials.

Molecular Detection of a Novel Fowl Adenovirus Serotype-4 (FadV-4) from an Outbreak of Hepatitis Hydropericardium Syndrome in Commercial Broiler Chickens in Egypt.

Hepatitis hydropericardium syndrome (HHS) is an acute infectious disease caused by fowl adenovirus serotype-4 (FAdV-4), which mainly affects broilers aged 4-5 wk. During the winter of January 2021, a 32-day-old broiler flock (Cobb-500) suffered from unusually high mortality (15%) in the Alexandria Governorate, Egypt. The chickens showed depression, ruffled feathers, and greenish diarrhea besides the typical pathologic features of suspected HHS involving flabby hearts, accumulation of a straw-colored fluid in the pericardial sacs, and pale, enlarged hemorrhagic and friable livers with necrotic foci. The kidneys exhibited edema with uric acid depositions. Histopathologic examination of bird livers naturally infected with HHS showed multifocal areas of necrosis, vascular changes, and basophilic intranuclear inclusion bodies (INIB) in the hepatocytes. Molecular identification of the causative agent was accomplished by PCR and sequence analysis of the hyper-variable regions of loop 1 of the hexon gene of fowl aviadenovirus. A pathogenic strain of the novel genotype-4 (FAdV-4) was demonstrated, closely similar to the Israeli strain IS/1905/2019, with an identity of 98%. This is the first report to identify FADV-4 in Egypt, prompting further studies to elucidate its epidemiologic role in all poultry sectors and associated economic losses to provide insights to its control and prevention.

Reporte de caso- Detección molecular de un nuevo serotipo 4 de adenovirus del pollo (FadV-4) de un brote del síndrome de hepatitis e hidropericardio en pollos de engorde comerciales en Egipto. El síndrome de hepatitis e hidropericardio (HHS) es una enfermedad infecciosa aguda causada por el adenovirus aviar serotipo-4 (FAdV-4), que afecta principalmente a pollos de engorde de 4 a 5 semanas de edad. Durante el invierno de enero de 2021, una parvada de pollos de engorde de 32 días (Cobb-500) sufrió una mortalidad inusualmente alta (15%) en la gobernación de Alejandría, en Egipto. Los pollos mostraban depresión, plumas erizadas y diarrea verdosa, además de las características lesiones típicas sugestivas del síndrome de hepatitis e hidropericardio, que involucraban corazones flácidos, acumulación de un líquido de color pajizo en los sacos pericárdicos e hígados pálidos, agrandados, hemorrágicos y friables con focos necróticos. Los riñones presentaban edema con depósitos de ácido úrico. El examen histopatológico de hígados de las aves naturalmente infectadas con el síndrome de hepatitis e hidropericardio mostraron áreas multifocales de necrosis, cambios vasculares y cuerpos de inclusión intranucleares basófilos en los hepatocitos. La identificación molecular del agente causal se logró mediante PCR y análisis de las secuencias de las regiones hipervariables del asa 1 del gene del hexón del aviadenovirus aviar. Se demostró la presencia de una cepa patógena del nuevo genotipo 4 (FAdV-4), muy similar a la cepa israelí IS/1905/2019, con una identidad del 98%. Este es el primer reporte que identifica el FADV-4 en Egipto, lo que motivó más estudios para dilucidar su papel epidemiológico en todos los sectores avícolas y las pérdidas económicas asociadas para proporcionar información sobre su control y prevención.

Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Avian Influenza Virus H9N2 HA Gene.

The H9N2 subtype of avian influenza A virus (aIAV) is circulating among birds worldwide, leading to severe economic losses. H9N2 cocirculation with other highly pathogenic aIAVs has the potential to contribute to the rise of new strains with pandemic potential. Therefore, rapid detection of H9 aIAVs infection is crucial to control virus spread. A qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of aIAV subtype H9N2 was developed. All results were compared to the gold standard (real-time reverse transcription polymerase chain reaction (RT-PCR)). The RT-RPA assay was designed to detect the hemagglutinin (HA) gene of H9N2 by testing three pairs of primers and a probe. A serial concentration between 106 and 100 EID50 (50% embryo infective dose)/mL was applied to calculate the analytical sensitivity. The H9 RT-RPA assay was highly sensitive as the lowest concentration point of a standard range at one EID50/mL was detected after 5 to 8 min. The H9N2 RT-RPA assay was highly specific as nucleic acid extracted from H9 negative samples and from other avian pathogens were not cross detected. The diagnostic sensitivity when testing clinical samples was 100% for RT-RPA and RT-PCR. In conclusion, H9N2 RT-RPA is a rapid sensitive and specific assay that easily operable in a portable device for field diagnosis of aIAV H9N2.

Simultaneous detection and differentiation by multiplex real time RT-PCR of highly pathogenic avian influenza subtype H5N1 classic (clade 2.2.1 proper) and escape mutant (clade 2.2.1 variant) lineages in Egypt.

BACKGROUND: The endemic status of highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 in Egypt continues to devastate the local poultry industry and poses a permanent threat for human health. Several genetically and antigenically distinct H5N1 lineages co-circulate in Egypt: Strains of clade 2.2.1 proper replicate mainly in backyard birds causing the bulk of human infections, while a variant lineage within 2.2.1 (2.2.1 v) appears to be perpetuated mainly in commercial poultry farms in Egypt. Viruses of the 2.2.1 v lineage represent drift variants escaping from conventional vaccine-induced immunity and some of these strains also escaped detection by commercial real time reverse transcriptase PCR (RT-qPCR) protocols due to mismatches in the primers/probe binding sites. RESULTS: We developed therefore a versatile, sensitive and lineage-specific multiplex RT-qPCR for detection and typing of H5N1 viruses in Egypt. Analytical characterization was carried out using 50 Egyptian HPAIV H5N1 strains isolated since 2006 and 45 other avian influenza viruses (AIV). A detection limit of 400 cRNA copies per ml sample matrix was found. Higher diagnostic sensitivity of the multiplex assay in comparison to other generic H5 or M-gene based RT-qPCR assays were found by examination of 63 swab samples from experimentally infected chickens and 50 AIV-positive swab samples from different host species in the field in Egypt. CONCLUSIONS: The new multiplex RT-qPCR assay could be useful for rapid high-throughput monitoring for the presence of HPAIV H5N1 in commercial poultry in Egypt. It may also aid in prospective epidemiological studies to further delineate and better control spread of HPAIV H5N1 in Egypt.