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.

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.

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.

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.

Detection of avian influenza virus: a comparative study of the in silico and in vitro performances of current RT-qPCR assays.

Avian influenza viruses (AIV) are negative sense RNA viruses posing a major threat to the poultry industry worldwide, with the potential to spread to mammals, including humans; hence, an accurate and rapid AIV diagnosis is essential. To date AIV detection relies on molecular methods, mainly RT-qPCR directed against AIV M gene segment. The evolution of AIV represents a relevant issue in diagnostic RT-qPCR due to possible mispriming and/or probe-binding failures resulting in false negative results. Consequently, RT-qPCR for AIV detection should be periodically re-assessed both in silico and in vitro. To this end, a specific workflow was developed to evaluate in silico the complementarity of primers and probes of four published RT-qPCR protocols to their target regions. The four assays and one commercially available kit for AIV detection were evaluated both for their analytical sensitivity using eight different viral dilution panels and for their diagnostic performances against clinical specimens of known infectious status. Differences were observed among the tests under evaluation, both in terms of analytical sensitivity and of diagnostic performances. This finding confirms the importance of continuously monitoring the primers and probes complementarity to their binding regions.

Genotyping and reassortment analysis of highly pathogenic avian influenza viruses H5N8 and H5N2 from Egypt reveals successive annual replacement of genotypes.

Highly pathogenic (HP) H5N1, clade 2.2.1, and low pathogenic avian influenza (LPAI) H9N2 viruses, G1-B lineage, are endemic in poultry in Egypt and have co-circulated for almost a decade. Surprisingly, no inter-subtypic reassortment events have been reported from the field during that time. After the introduction of HPAIV H5N8, clade 2.3.4.4b, in Egyptian poultry in 2016, suddenly HP H5N2 reassortants with H9N2 viruses emerged. The current analyses focussed on studying 32 duck flocks, 4 broiler chicken flocks, and 1 turkey flock, suffering from respiratory manifestations with moderate to high mortality reared in two Egyptian governorates during 2019. Real-time RT-PCR substantiated the presence of HP H5N8 in 21 of the 37 investigated flocks with mixed infection of H9N2 in two of them. HP H5N1 was not detected. Full hemagglutinin (HA) sequencing of 10 samples with full-genome sequencing of three of them revealed presence of a single genotype. Very few substituting mutations in the HA protein were detected versus previous Egyptian HA sequences of that clade. Interestingly, amino acid substitutions in the Matrix (M2) and the Neuraminidase (NA) proteins associated with conferring both Amantadine and Oseltamivir resistance were present. Systematic reassortment analysis of all publicly available Egyptian whole genome sequences of HP H5N8 (n = 23), reassortant HP H5N2 (n = 2) and LP H9N2 (n = 53) viruses revealed presence of at least seven different genotypes of HPAI H5Nx viruses of clade 2.3.4.4b in Egypt since 2016. For H9N2 viruses, at least three genotypes were distinguishable. Heat mapping and tanglegram analyses suggested that several internal gene segments in both HP H5Nx and H9N2 viruses originated from avian influenza viruses circulating in wild bird species in Egypt. Based on the limited set of whole genome sequences available, annual replacement patterns of HP H5Nx genotypes emerged and suggested selective advantages of certain genotypes since 2016.

Disentangling the role of Africa in the global spread of H5 highly pathogenic avian influenza.

The role of Africa in the dynamics of the global spread of a zoonotic and economically-important virus, such as the highly pathogenic avian influenza (HPAI) H5Nx of the Gs/GD lineage, remains unexplored. Here we characterise the spatiotemporal patterns of virus diffusion during three HPAI H5Nx intercontinental epidemic waves and demonstrate that Africa mainly acted as an ecological sink of the HPAI H5Nx viruses. A joint analysis of host dynamics and continuous spatial diffusion indicates that poultry trade as well as wild bird migrations have contributed to the virus spreading into Africa, with West Africa acting as a crucial hotspot for virus introduction and dissemination into the continent. We demonstrate varying paths of avian influenza incursions into Africa as well as virus spread within Africa over time, which reveal that virus expansion is a complex phenomenon, shaped by an intricate interplay between avian host ecology, virus characteristics and environmental variables.

Efficacy of Clade 2.3.2 H5-Recombinant Baculovirus Vaccine in Protecting Muscovy and Pekin Ducks from Clade 2.3.4.4 H5N8 Highly Pathogenic Avian Influenza Infection.

In late 2016, a highly pathogenic avian influenza (HPAI) virus subtype H5N8 clade 2.3.4.4 was reported in Egypt in migratory birds; subsequently, the virus spread to backyard and commercial poultry in several Egyptian governorates, causing severe economic losses to the poultry industry. Here, a recombinant subunit commercial H5 vaccine prepared from the clade 2.3.2 H5 segment on baculovirus was evaluated in Pekin ducks (Anasplatyrhynchos domesticus) and Muscovy ducks (Cairina moschata) in Biosafety Level 3 isolators by using two vaccination regimes: either a single dose on day 10 and a challenge on day 31 or a double dose on days 10 and 28 and a challenge on day 49. The protection parameters were evaluated after experimental infection with the Egyptian HPAI H5N8 isolate clade 2.3.4.4b (A/common-coot/Egypt/CA285/2016) based on mortality rate, clinical signs, gross lesions, seroconversion, virus shedding, and histopathologic changes. In the single-dose vaccination regime, the mortality rate in Muscovy and Pekin ducks was 10% and 0% vs. 40% and 0% in nonvaccinated challenged ducks, respectively. In the double-dose vaccination regime, the mortality rates in Muscovy and Pekin ducks were 0% and 0% vs. 60% and 40% in nonvaccinated challenged ducks, respectively. Muscovy ducks developed more severe clinical signs and gross lesions than Pekin ducks. In addition, tracheal viral shedding in challenged Muscovy ducks, in the single-dose vaccination regime, was 50%, 22%, and 0% at 3, 5, and 7 days postchallenge (DPC), respectively, and was 0% in all Pekin ducks vs. 100% in all challenged nonvaccinated Muscovy and Pekin ducks at 3, 5, and 7 DPC. The viral shedding in challenged Muscovy and Pekin ducks, in the double-dose vaccination regime, was 0% at 3, 5, and 7 DPC vs. 100% in nonvaccinated challenged Muscovy and Pekin ducks, respectively. The results of this study indicate that the H5 baculovirus-based vaccine can be used in ducks with better vaccination regime based on double-dose vaccination at 10 and 28 days of age. In addition, they highlight the need to evaluate the efficacy of currently used commercial vaccines against challenge with the newly emerged HPAI H5N8 clade 2.3.4.4 in the field in Egypt to ensure proper control strategy in ducks.

Eficacia de una vacuna desarrollada con un baculovirus recombinante subtipo H5 clado 2.3.2 en la protección de patos reales y Pekín contra la infección con virus de la influenza aviar de alta patogenicidad subtipo H5N8, clado 2.3.4.4. A finales del año 2016, se reportó en aves migratorias en Egipto la presencia del virus de la influenza aviar de alta patogenicidad subtipo H5N8, clado 2.3.4.4. Posteriormente, el virus se propagó en aves de traspatio y comerciales de varias provincias egipcias, causando graves pérdidas económicas a la industria avícola. En este trabajo, una vacuna subunitaria recombinante comercial con el subtipo H5 preparada a partir del segmento H5 del clado 2.3.2 expresado en baculovirus se evaluó en patos de Pekín y reales en unidades de aislamiento con nivel de bioseguridad 3 utilizando dos esquemas de vacunación: una dosis única en el día 10 y un desafío el día 31; o un esquema con doble dosis en los días 10 y 28 y con un desafío en el día 49. Los parámetros de protección se evaluaron después de la infección experimental con el aislamiento del virus de alta patogenicidad H5N8, clado 2.3.4.4b de Egipto (A/focha común/Egipto/ CA285/2016) con base en la tasa de mortalidad, signos clínicos, lesiones macroscópicas, seroconversión, eliminación del virus y cambios histopatológicos. Los resultados revelaron que la tasa de mortalidad en patos reales y Pekín, en un régimen de vacunación con dosis única fue de 10% y 0%, respectivamente en comparación con 40% y 0% en patos no vacunados y desafiados, respectivamente. En los patos reales y Pekín, con un esquema de vacunación con dosis doble, la tasa de mortalidad fue del 0% en comparación con 60% y 40% en los patos no vacunados y desafiados, respectivamente. Los patos reales desarrollaron signos clínicos y lesiones más severos en comparación con los patos Pekín. Además, la eliminación viral a partir de la tráquea en patos reales desafiados y con un esquema de vacunación de dosis única, fue del 50%, 22% y 0% a los 3, 5 y 7 días posteriores al desafío, respectivamente, y fue del 0% en todos los patos Pekín en comparación con el 100% en todos los patos reales y Pekín no vacunados y desafiados a los 3, 5 y 7 días después del desafío. La eliminación viral en los patos reales y Pekín desafiados, con un esquema de vacunación de dosis doble, fue de 0% a los tres, cinco y siete días después del desafío en comparación con el 100% en los patos reales y Pekín no vacunados y desafiados, respectivamente. Los resultados de este estudio indican que la vacuna basada en el baculovirus H5 se puede usar en patos con un mejor esquema de vacunación basado en la vacunación con dosis doble a los 10 y 28 días de edad. Además, se resalta la necesidad de evaluar la eficacia de las vacunas comerciales utilizadas actualmente contra el desafío con el nuevo virus de alta patogenicidad H5N8 clado 2.3.4.4 en el campo en Egipto para garantizar una estrategia de control adecuada en patos.

Molecular pathogenic and host range determinants of reassortant Egyptian low pathogenic avian influenza H9N2 viruses from backyard chicken.

Since the introduction of H9N2 low pathogenic avian influenza virus in Egypt, it became an endemic disease causing considerable economic losses in different poultry sectors especially in the presence of other secondary bacterial and viral infections. The H9N2 viruses in Egypt are in continuous evolution that needs deep analysis for their evolution pattern based on the genetic constitutions of the pathogenic determinant genes (HA, PB2, PB1, PA, and NS). In this work, samples were collected from the backyard chickens from 3 Egyptian governorates. Five selected viruses were sequenced and analyzed for the hemagglutinin gene which showed genetic relatedness to the Asian G1 lineage group B, similar to the circulating H9N2 viruses in Egypt since 2013. The sequence for PB2, PB1, PA, HA and NS genes of the selected five viruses indicate a natural re-assortment event with recent Eurasian subtypes and similar to Egyptian H9N2 virus isolated from pigeon in Egypt during 2014. The Egyptian viruses of our study possess amino acids signatures including S42, V127, L550, L672 and V504 in the internal genes NS1, PA, and PB2, of respectively of an impact on virus transmission and replication. This work indicates that the H9N2 is in continuous evolution with alarming to the reassortment occurrence.

Isolation of a Novel Reassortant Highly Pathogenic Avian Influenza (H5N2) Virus in Egypt.

Highly pathogenic avian influenza (HPAI) H5N1 and H5N8 have become endemic among domestic poultry in Egypt since 2006 and 2016, respectively. In parallel, the low pathogenic avian influenza H9N2 virus has been endemic since 2010. Despite the continuous circulation of these subtypes for several years, no natural reassortant has been detected so far among the domestic poultry population in Egypt. In this study, the HPAI (H5N2) virus was isolated from a commercial duck farm, giving evidence of the emergence of the first natural reassortment event in domestic poultry in Egypt. The virus was derived as a result of genetic reassortment between avian influenza viruses of H5N8 and H9N2 subtypes circulating in Egypt. The exchange of the neuraminidase segment and high number of acquired mutations might be associated with an alteration in the biological propensities of this virus.

Combined H5ND inactivated vaccine protects chickens against challenge by different clades of highly pathogenic avian influenza viruses subtype H5 and virulent Newcastle disease virus.

AIM: The aim of the current study was to evaluate the efficacy of a trivalent-inactivated oil-emulsion vaccine against challenge by different clades highly pathogenic avian influenza (HPAI) viruses including HPAI-H5N8 and the virulent genotype VII Newcastle disease virus (NDV) (vNDV). MATERIALS AND METHODS: The vaccine studied herein is composed of reassortant AI viruses rgA/Chicken/Egypt/ME1010/2016 (clade 2.2.1.1), H5N1 rgA/Chicken/Egypt/RG-173CAL/2017 (clade 2.2.1.2), and "NDV" (LaSota NDV/CK/Egypt/11478AF/11); all used at a concentration of 108 EID50/bird and mixed with Montanide-ISA70 oil adjuvant. Two-week-old specific pathogen free (SPF) chickens were immunized subcutaneously with 0.5 ml of the vaccine, and hemagglutination inhibition (HI) antibody titers were monitored weekly. The intranasal challenge was conducted 4 weeks post-vaccination (PV) using 106 EID50/0.1 ml of the different virulent HPAI-H5N1 viruses representing clades 2.2.1, 2.2.1.1, 2.2.1.2, 2.3.4.4b-H5N8, and the vNDV. RESULTS: The vaccine induced HI antibody titers of >6log2 against both H5N1 and NDV viruses at 2 weeks PV. Clinical protection against all HPAI H5N1 viruses and vNDV was 100%, except for HPAI H5N1 clade-2.2.1 and HPAI H5N8 clade-2.3.4.4b viruses that showed 93.3% protection. Challenged SPF chickens showed significant decreases in the virus shedding titers up to <3log10 compared to challenge control chickens. No virus shedding was detected 6 "days post-challenge" in all vaccinated challenged groups. CONCLUSION: Our results indicate that the trivalent H5ND vaccine provides significant clinical protection against different clades of the HPAI viruses including the newly emerging H5N8 HPAI virus. Availability of such potent multivalent oil-emulsion vaccine offers an effective tool against HPAI control in endemic countries and promises simpler vaccination programs.

Efficacy of Bivalent Inactivated Vaccine Containing Insect Cell-Expressed Avian Influenza H5 and Egg-Based Newcastle Disease Virus (NDV) Against Dual Infection with Highly Pathogenic H5N1 and Velogenic NDV in Chickens.

The genetic evolution of highly pathogenic avian influenza (HPAI) in Egypt has developed a new clade H5N1 (2.2.1.2) since 2014. Meanwhile, the new avian influenza virus (AIV) clade mutually with the velogenic Newcastle disease virus (NDV) isolate of genotype VII in Egypt (genotype VII) has resulted in severe economic losses in the broiler industry. An inactivated bivalent vaccine containing H5 (belonging to H5N1 clade 2.3.2) recombinant baculovirus expressed by insect cell (recH5) and egg-based NDV LaSota strain (recH5/NDV vaccine) was evaluated for protection against the challenge of dual HPAIV H5N1 clade 2.2.1.2 and vNDV infection in commercial broiler chickens. Vaccination was performed when chickens were 10 days old, and then birds of the respective groups were challenged with 106 50% egg infective dose per chicken of each virus in 100 µl of allantoic fluid via the intranasal route at 21 days postvaccination in a single or sequential infection of both viruses. Results showed that the recH5/NDV vaccine was able to protect chickens against single or dual challenges of both viruses ranging up to 90%-100%. Unvaccinated chickens have demonstrated 100% mortalities to a single virus challenge. Vaccinated chickens showed significant decreases in both viruses, shedding titers up to <2 log 10 after challenge in comparison with unvaccinated ones. Cessation of viral shedding was obtained at 7 to 10 days postchallenge. The vaccinated chickens showed high hemagglutination inhibition antibody titers >6 log 2 against both H5N1 and NDV antigens at 2 wk postvaccination. The single vaccination of bivalent inactivated recH5-NDV vaccine at 10 days old in commercial chickens has provided significant clinical protective immunity against single or dual challenge with HPAI-H5N1clade 2.2.1.2 and vNDV-genotype VII.

Eficacia en pollos de una vacuna inactivada bivalente que contiene la proteína H5 del virus de influenza aviar H5 expresada en células de insecto y el virus de la enfermedad de Newcastle replicado en embrión de pollo contra la infección dual con un virus de influenza aviar H5N1 altamente patogénico y un virus de la enfermedad de Newcastle velogénico. La evolución genética de la influenza aviar altamente patógena (HPAI) en Egipto ha desarrollado un nuevo clado H5N1 (2.2.1.2) desde el año 2014. Mientras tanto, el nuevo clado del virus de la influenza aviar (AIV) junto con aislados del virus de la enfermedad de Newcastle velogénicos de genotipo VII en Egipto ha provocado graves pérdidas económicas para la industria de pollos de engorde. Una vacuna bivalente inactivada que contiene la proteína H5 (perteneciente al clado H5N1 2.3.2) expresada en un baculovirus recombinante y replicado células de insecto (recH5) junto con la cepa LaSota del virus de Newcastle replicada en embriones de pollo (vacuna recH5/NDV) fue evaluada en la protección contra el desafío doble con un virus de influenza aviar de alta patogenicidad H5N1 clado 2.2.1.2 y contra un virus virulento de la enfermedad de Newcastle en pollos de engorde comerciales. La vacunación se realizó cuando los pollos tenían diez días de edad y luego las aves de los grupos respectivos fueron desafiadas con 106 dosis infectivas para embrión de pollo al 50% por pollo de cada virus en 100 µl de líquido alantoideo a través de la vía intranasal a los 21 días posteriores a la vacunación en una sola infección o con infección secuencial con ambos virus. Los resultados mostraron que la vacuna recH5/NDV fue capaz de proteger a los pollos contra los desafíos simples o dobles con ambos virus con un rango que va del 90% al 100%. Los pollos no vacunados mostraron 100% de mortalidad ante el desafío simple con un solo virus. Los pollos vacunados mostraron disminuciones significativas en la eliminación de ambos virus, arrojando títulos menores de 2 log10 después del desafío, en comparación con las aves no vacunadas. El cese de la propagación viral se observó de los siete a los diez días posteriores al desafío. Los pollos vacunados mostraron altos títulos de anticuerpos por inhibición de la hemaglutinación con títulos mayores de 6log2 contra los antígenos H5N1 y NDV a las dos semanas posteriores a la vacunación. La inmunización única con la vacuna recH5/NDV inactivada bivalente a los 10 días de edad en pollos comerciales proporcionó una inmunidad protectora clínica significativa contra el desafío simple o doble con un virus de influenza aviar H5N1clado 2.2.1.2 y por un virus virulento de la enfermedad de Newcastle genotipo VII.

Full genome sequences of chicken anemia virus demonstrate mutations associated with pathogenicity in two different field isolates in Egypt.

Chicken anemia virus (CAV) is an important pathogen associated with immunosuppression in chicken. In this study, out of samples collected from 115 commercial poultry farms, 12 samples were CAV positive by PCR. Partial sequence and phylogenetic analysis of VP1 gene revealed that the detected viruses were clustered to genotype I (n = 3) and genotype II (n = 9). Motifs of both low (E144) and high pathogenic strains (T89, I125, Q141) were found in the three viruses of genotype I. Whereas genotype II viruses demonstrated the characteristic motifs of highly pathogenic strains (I75, T89, I125, Q141, and Q144). Three isolates representative of both genotypes (CAV/CA1, CAV/GZ1 and CAV/SK4) were selected for full genome sequencing and results revealed that the VP2 gene had two substitutions at V153 and E 175, while VP3 gene had only one substitution at C118. To evaluate virus pathogenicity, two isolates from each genotype (CAV/SK4 of genotype I and CAV/CA1 of genotype II) were intramuscularly inoculated in two groups of one-day-old specific pathogen free chicks. Eighteen days post inoculation, PCR detected CAV in 75 and 90% of chicks in group I and II; respectively. Mortalities in inoculated chicks were 5 and 20% and packed cell volume values were 0.21 and 0.19; respectively. CAV/CA1 and CAV/SK4 isolates showed pathogenic evidences at the level of genetic (Q141 and 394Q) with variable degree of virulence. In conclusion, the study reports the circulation of at least two genotypes of CAV among chicken population with mutation associated with pathogenicity.

Molecular characterization of full fusion protein (F) of Newcastle disease virus genotype VIId isolated from Egypt during 2012-2016.

AIM: The aim of this work was to study the full F gene sequence of Newcastle disease virus (NDV) in regard to pathotyping and genotyping and to study the evolution of this NDV in Egypt. MATERIALS AND METHODS: The present study was conducted using samples from seven suspected NDV flocks of vaccinated chickens during 2012-2016 from six governorates in Egypt. The NDV was successfully isolated from pathological specimens through inoculation in specific pathogen-free embryonated chicken eggs. RESULTS: Pathogenicity of the NDV isolates has been estimated through intracerebral pathogenicity index and ranged from 1.66 to 1.73 which indicates the velogenic type of NDV isolates. Pathotyping and genotyping of these isolates were done through sequencing of full-length F gene. Results indicated that the seven NDV isolates showed characteristic cleavage site motif (112RRQKRF117) for the velogenic strains of NDV. Phylogenetic analysis of the F gene clustered these isolates within Group I of genotype VIId within Israeli strains NDV/IS/2015, NDV-Ch/SD883, and most of the Middle East strains. Six of seven sequenced isolates have six potential N-linked glycosylation sites. The neutralization epitope on the five antigenic sites of fusion is conserved in all Egyptian strains of this study except NDV-KFR-B7-2012 which has a substitution at D 170 N in epitope A4. In all our strains, 10 cysteine residues are recorded, except one loss of cysteine at residue 370 in both NDV-EG-35-2014 and NDV-GHB-328F-2016. CONCLUSION: All viruses in this study have 52 amino acid substitutions within fusion gene in compared with Lasota strain that reveals importance for its antigenic and structural function. The present work highlights the important need to sequence F gene of NDV genotype VIId to investigate the evolution of this NDV in Egypt.

Potential Biological and Climatic Factors That Influence the Incidence and Persistence of Highly Pathogenic H5N1 Avian Influenza Virus in Egypt.

Highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 is endemic in poultry in Egypt where the highest number of human infections worldwide was reported. During the last 12 years the Egyptian A/H5N1 evolved into several genotypes. In 2007-2014 vaccinated poultry suffered from antigenic drift variants of clade 2.2.1.1 and in 2014/2015 an unprecedented upsurge of A/H5N1 clade 2.2.1.2 occurred in poultry and humans. Factors contributing to the endemicity or re-emergence of A/H5N1 in poultry in Egypt remain unclear. Here, three potential factors were studied: climatic factors (temperature, relative humidity, and wind speed), biological fitness in vitro, and pathogenicity in domestic Pekin and Muscovy ducks. Statistical analyses using negative binomial regression models indicated that ambient temperature in winter months influenced the spread of A/H5N1 in different geographic areas analyzed in this study. In vitro, at 4 and 56°C 2.2.1.1 and recent 2.2.1.2 viruses were more stable than other viruses used in this study. Further, Pekin ducks were more resistant than Muscovy ducks and the viruses were excreted for up to 2 weeks post-infection assuming a strong role as a reservoir. Taken together, ambient temperature in winter months potentially contributes to increasing outbreaks in some regions in Egypt. Heat stability of clade 2.2.1.1 and recent 2.2.1.2 viruses probably favors their persistence at elevated temperatures. Importantly, asymptomatically infected Pekin ducks may play an important role in the spread of avian and human-like A/H5N1 in Egypt. Therefore, control measures including targeted surveillance and culling of silently infected Pekin ducks should be considered.

In silico thermodynamic stability of mammalian adaptation and virulence determinants in polymerase complex proteins of H9N2 virus.

The polymerase complex proteins (PB2, PB1, and PA) are responsible primarily for the replication of avian influenza virus and play an important role in virus virulence, mammalian adaptation, and interspecies transmission. In this study; eight Egyptian LPAI-H9N2 viruses isolated from apparent healthy chickens and quails from 2014 to 2016. Characterization of complete nucleotide sequences, phylogenetic and mutation analysis were carried out. The measurement of thermodynamic stability of the H9N2 polymerase protein in comparison to human H3N2 and H1N1 proteins was carried out using in silico method. Phylogenetic analysis of these viruses revealed a close relationship to viruses isolated from neighboring Middle Eastern countries with an average of 96-99% homology. They are sharing the common ancestor A/quail/Hong Kong/G1/1997 (G1-Like) without any evidence for genetic reassortment. In addition, eight markers related to virulence were identified, including the combination of 627V and 391E in the PB2 gene with full-length PB1-F2 and PA-X proteins were observed in all viruses and the substitution N66S in PB1-F2 which suggest increasing virus virulence. Moreover, six markers that may affect the virus replication and transmission in mammalian hosts were identified. Five mutations related to mammalian adaptation show a structural stabilizing effect on LPAI-H9N2 polymerase complex protein according to the free-energy change (ΔΔG). Three out of those six adaptive mutations shown to increase polymerase complex protein stability were found in Egyptian LPAI-H9N2 viruses similar to Human H3N2 and H1N1 (661 in PB2, 225 and 409 in PA genes). Our results suggested that the stabilizing mutations in the polymerase complex protein have likely affected the protein structure and induced favorable conditions for avian virus replication and transmission in mammalian hosts. Indeed, the study reports the mutational analysis of the circulating LPAI-H9N2 strains in Egypt.

Multiple introductions of reassorted highly pathogenic avian influenza viruses (H5N8) clade 2.3.4.4b causing outbreaks in wild birds and poultry in Egypt.

Recently, an increased incidence of outbreaks of highly pathogenic avian influenza (HPAI) H5N8 in poultry linked to infected migratory birds has been reported from different European, Asian and African countries. In Egypt, incursion of HPAI H5N8 virus of clade 2.3.4.4b has been recently registered. Full genomic characterization of 3 virus isolates from wild birds and poultry (backyard and commercial farm sectors) showed high nucleotide similarity among the HA, NA, M, and NS gene segments of the three Egyptian HPAI H5N8 viruses, indicating that they are descendants of a common ancestral virus. However, the analyzed Egyptian H5N8 viruses revealed distinct genotypes involving different origins of the PB2, PB1, PA and/or NP segments. In genotype-1 represented by strain A/common-coot/Egypt/CA285/2016 the PB2 and NP segments showed closest relationship to H5N6 and H6N2 viruses, recently detected in Italy. The second is replacement of PB1 and NP genes A novel reassortant, represented by strain A/duck/Egypt/SS19/2017, showed an exchange of PB1 and NP genes which might have originated from H6N8 or H1N1 and H6N2 viruses. Finally, replacement of PA and NP genes characterized strain A/duck/Egypt/F446/2017. Bayesian phylogeographic analyses revealed that Egyptian H5N8 viruses are highly likely derived from Russian 2016 HPAI H5N8 virus (A/great_crested_grebe/Uvs-Nuur_Lake/341/2016 (H5N8)) and the reassortment likely occurred before incursion to Egypt.

Reactivity and sensitivity of commercially available influenza rapid diagnostic tests in Japan.

Seasonal influenza virus routinely causes epidemic infections throughout the world. Sporadic infections by H5N1, H5N6, and H7N9 viruses are also reported. To treat patients suffering from such viral infections, broadly reactive and highly sensitive influenza rapid diagnostic tests (IRDTs) are required. Here, we examined the reactivity and sensitivity of 25 IRDTs available in Japan for the detection of seasonal H1N1pdm09, H3N2, and type B viruses, as well as highly pathogenic H5 and H7 viruses. All of the IRDTs tested detected the seasonal viruses and H5 and H7 viruses albeit with different sensitivities. Several IRDTs detected the H5 and H7 viruses and the seasonal viruses with similar (high) sensitivity.