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.

Pathogenicity of the Egyptian A/H5N1 avian influenza viruses in chickens.

Long-term circulation of highly pathogenic avian influenza H5N1 viruses of clade 2.2.1 in Egyptian poultry since February 2006 resulted in the evolution of two distinct clades: 2.2.1.1 represents antigenic-drift variants isolated from vaccinated poultry and 2.2.1.2 that caused the newest upsurge in birds and humans in 2014/2015. In the present study, nine isolates were collected from chickens, ducks and turkeys representing the commercial and backyard sectors during the period 2009-2015. The subtyping was confirmed by hemagglutination inhibition (HI) test, RT-qPCR and sequence analysis. The Mean Death Time (MDT) and Intravenous Pathogenicity Index (IVPI) for all isolates were determined. Sequence analysis of the HA gene sequences of these viruses revealed that two viruses belonged to clade 2.2.1.1 and the rest were clade 2.2.1.2. Antigenic characterisation of the viruses supported the results of the phylogenetic analysis. The MDT of the isolates ranged from 18 to 72 h and the IVPI values ranged from 2.3 to 2.9; viruses of the 2.2.1.1 clade were less virulent than those of the 2.2.1.2 clade. In addition, clade-specific polymorphism in the HA cleavage site was observed. These findings indicate the high and variable pathogenicity of H5N1 viruses of different clades and host-origin in Egypt. The upsurge of outbreaks in poultry in 2014/2015 was probably not due to a shift in virulence from earlier viruses.

A brief summary of the epidemiology and genetic relatedness of avian influenza H9N2 virus in birds and mammals in the Middle East and North Africa.

H9N2 is the most widespread avian influenza virus subtype in poultry worldwide. It infects a broad spectrum of host species including birds and mammals. Infections in poultry and humans vary from silent to fatal. Importantly, all AIV, which are fatal in humans (e.g. H5N1, H7N9) acquired their 'internal' gene segments from H9N2 viruses. Although H9N2 is endemic in the Middle East (ME) and North Africa since the late 1990s, little is known about its epidemiology and genetics on a regional level. In this review, we summarised the epidemiological situation of H9N2 in poultry and mammals in Iran, Iraq, Kuwait, Qatar, United Arab Emirates, Oman, Bahrain, Yemen, Saudi Arabia, Jordan, Palestine, Israel, Syria, Lebanon, Turkey, Egypt, Sudan, Libya, Tunisia, Algeria and Morocco. The virus has been isolated from humans in Egypt and serosurveys indicated widespread infection particularly among poultry workers and pigs in some countries. Some isolates replicated well in experimentally inoculated dogs, mice, hamsters and ferrets. Insufficient protection of immunised poultry was frequently reported most likely due to concurrent viral or bacterial infections and antigenic drift of the field viruses from outdated vaccine strains. Genetic analysis indicated several distinct phylogroups including a panzootic genotype in the Asian and African parts of the ME, which may be useful for the development of vaccines. The extensive circulation of H9N2 for about 20 years in this region where the H5N1 virus is also endemic in some countries, poses a serious public health threat. Regional surveillance and control strategy are highly recommended.

Evolutionary features of influenza A/H5N1 virus populations in Egypt: poultry and human health implications.

Since 2006, in Egypt, highly pathogenic avian influenza virus (HPAIV) H5N1 has established endemic status in poultry. Bayesian evolutionary analysis sampling trees suggested an introduction date in the third quarter of 2005. Evolutionary dynamics using Bayesian analysis showed that H5N1 viruses of clade 2.2.1.1 evolved at higher rates than those of clade 2.2.1.2. Bayesian skyline plot analysis of the HA gene of 840 and NA gene of 401 Egyptian H5N1 viruses from 2006-2015 identified two waves of viral population expansion correlating with the stepwise emergence of the 2.2.1.1 variant lineage in 2008 and with the newly emerging 2.2.1.2 cluster in late 2014. H5N1 infections in human hosts in 2014-2015 were statistically linked to a contemporary poultry outbreak.

Emergence of a novel cluster of influenza A(H5N1) virus clade 2.2.1.2 with putative human health impact in Egypt, 2014/15.

A distinct cluster of highly pathogenic avian influenzaviruses of subtype A(H5N1) has been found to emergewithin clade 2.2.1.2 in poultry in Egypt since summer2014 and appears to have quickly become predominant.Viruses of this cluster may be associated withincreased incidence of human influenza A(H5N1) infectionsin Egypt over the last months.

Prevalence and control of H7 avian influenza viruses in birds and humans.

The H7 subtype HA gene has been found in combination with all nine NA subtype genes. Most exhibit low pathogenicity and only rarely high pathogenicity in poultry (and humans). During the past few years infections of poultry and humans with H7 subtypes have increased markedly. This review summarizes the emergence of avian influenza virus H7 subtypes in birds and humans, and the possibilities of its control in poultry. All H7Nx combinations were reported from wild birds, the natural reservoir of the virus. Geographically, the most prevalent subtype is H7N7, which is endemic in wild birds in Europe and was frequently reported in domestic poultry, whereas subtype H7N3 is mostly isolated from the Americas. In humans, mild to fatal infections were caused by subtypes H7N2, H7N3, H7N7 and H7N9. While infections of humans have been associated mostly with exposure to domestic poultry, infections of poultry have been linked to wild birds or live-bird markets. Generally, depopulation of infected poultry was the main control tool; however, inactivated vaccines were also used. In contrast to recent cases caused by subtype H7N9, human infections were usually self-limiting and rarely required antiviral medication. Close genetic and antigenic relatedness of H7 viruses of different origins may be helpful in development of universal vaccines and diagnostics for both animals and humans. Due to the wide spread of H7 viruses and their zoonotic importance more research is required to better understand the epidemiology, pathobiology and virulence determinants of these viruses and to develop improved control tools.

In vitro inactivation of two Egyptian A/H5N1 viruses by four commercial chemical disinfectants.

The highly pathogenic H5N1 avian influenza virus (A/H5N1) devastated the poultry industry and posed a serious health threat. Cleaning and disinfection are essential parts of preventative and postoutbreak management of A/H5N1 infections in poultry. In this preliminary study, we used suspension and carrier tests to evaluate the impact of concentration, time of exposure, surface porosity, and organic matter on the ability of four commercial chemical disinfectants to inactivate two A/H5N1 viruses of clade 2.2.1 isolated in 2006 and 2010 from broiler flocks in Egypt. Viruses were incubated with 0.5%, 1%, and 2% of formalin, glutaraldehyde, TH4, and Virkon S for 15, 30, 60, and 120 min at room temperature (22 +/- 2 C). In suspension tests, in the absence of organic matter, all disinfectants, at each concentration, except Virkon S 0.5%, effectively inactivated virus suspensions after a 15-min exposure time. In the presence of organic matter, the use of low concentrations of formalin (0.5%), glutaraldehyde (0.5%), or Virkon S (0.5%) was not sufficient to inactivate the viruses after 15 min. In gauze carrier tests, only formalin at any concentration for 15 min was sufficient to inactivate the viruses, whereas different concentrations or exposure times were required for glutaraldehyde (0.5% for 60 min), TH4 (0.5% for 30 min), and Virkon S (0.5% for 60 min or 1% for 30 min). In wood carrier tests, total inactivation of the virus was obtained at concentrations of 0.5% for 30 min (formalin and TH4) or 60 min (glutaraldehyde and Virkon S). This study emphasizes the need to use high concentrations of and/or extended time of exposure to disinfectants for efficient inactivation of A/H5N1, particularly in the presence of organic matter or different surfaces, which are common in poultry operations. In addition, it seemed that the virus isolated in 2010 was more resistant to disinfectants than the isolate from 2006 when wood was used as a carrier.

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.

Detection of A/H5N1 virus from asymptomatic native ducks in mid-summer in Egypt.

In spite of all the efforts to control H5N1 in Egypt, the virus still circulates endemically, causing significant economic losses in the poultry industry and endangering human health. This study aimed to elucidate the role of clinically healthy ducks in perpetuation of H5N1 virus in Egypt in mid-summer, when the disease prevalence is at its lowest level. A total of 927 cloacal swabs collected from 111 household and 71 commercial asymptomatic duck flocks were screened by using a real-time reverse transcription polymerase chain reaction. Only five scavenging ducks from a native breed in three flocks were found infected with H5N1 virus. This study indicates that H5N1 virus can persist in free-range ducks in hot weather, in contrast to their counterparts confined in household or commercial settings. Surveillance to identify other potential reservoirs is essential.

Development of real-time polymerase chain reaction assay for detection of Ornithobacterium rhinotracheale in poultry.

Ornithobacterium rhinotracheale (ORT) is an emerging bacterium causing severe economic losses in poultry mostly due to respiratory and locomotory disturbances. Due to the fastidious nature of the organism, ORT is often overgrown by faster-growing commensal and pathogenic bacteria. In this study we developed a real-time polymerase chain reaction (qPCR) assay for rapid and sensitive detection of ORT in samples collected from chickens and turkeys. The qPCR assay developed was able to detect 17 reference strains of ORT (serotypes A to Q) tested in this study, and no false-positive results were obtained from other organisms associated with respiratory tract infections. The qPCR assay was 100 times more sensitive than the modified conventional PCR. Using tenfold serial dilutions of the recombinant plasmid DNA containing the target gene fragment, the detection limit of the qPCR was estimated to be > or = 100 plasmid copies per reaction. Out of 42 examined poultry flocks, 26 cases were tested positive by both assays. The qPCR assay reduces turnaround time to about 2 hr, two times faster than the modified conventional PCR.

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.

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.

Persistence of Histomonas meleagridis in or on materials used in poultry houses.

Histomonas meleagridis is the causative agent of blackhead disease or histomonosis in turkeys, and previous research suggests that this parasite survives poorly outside of hosts except within heterakid nematodes. However, we investigated the viability of H. meleagridis in or on several artificially contaminated materials kept at ambient room temperature (22 +/- 2 C) to mimic the situation in the field. The protozoan survived for up to 1 hr on wood, rubber, and metal; up to 3 hr on egg-tray cartons, egg shells, and bricks; up to 6 hr on straw, turkey feathers, and feed; and up to 9 hr in nonchlorinated tap water and fecal matter. Therefore, contaminated water, fresh fecal matter, or both could play a role in transmission of the parasite within and among poultry houses rather than other materials tested in this study.

An overview of the epidemic of highly pathogenic H5N1 avian influenza virus in Egypt: epidemiology and control challenges.

Emergence of the highly pathogenic avian influenza (HPAI) H5N1 virus in Egypt in mid-February 2006 caused significant losses for the poultry industry and constituted a potential threat to public health. Since late 2007, there has been increasing evidence that stable lineages of H5N1 viruses are being established in chickens and humans in Egypt. The virus has been detected in wild, feral and zoo birds and recently was found in donkeys and pigs. Most of the outbreaks in poultry and humans occurred in the highly populated Nile delta. The temporal pattern of the virus has changed since 2009 with outbreaks now occurring in the warmer months of the year. Challenges to control of endemic disease in Egypt are discussed. For the foreseeable future, unless a global collaboration exists, HPAI H5N1 virus in Egypt will continue to compromise the poultry industry, endanger public health and pose a serious pandemic threat.

Modified H5 real-time reverse transcriptase-PCR oligonucleotides for detection of divergent avian influenza H5N1 viruses in Egypt.

The efforts exerted to prevent circulation of highly pathogenic avian influenza (HPAI) H5N1 virus in birds are the best way to prevent the emergence of a new virus subtype with pandemic potential. Despite the blanket vaccination strategy against HPAI H5N1 in Egypt, continuous circulation of the virus in poultry has increased since late 2007 as a result of the presence of genetic and antigenic distinct variant strains that have escaped during the immune response of vaccinated birds. Although the suspected poultry flocks have had signs and lesions commonly seen in HPAI H5N1-infected birds, escape of variant strains from detection by real-time reverse transcriptase-PCR (RRT-PCR) was observed. Sequence analysis of these variants revealed multiple single nucleotide substitutions in the primers and probe target sequences of the H5 gene by real-time RT-PCR. This study describes the results of RRT-PCR, modified from an existing protocol with regard to the detection of the partial H5 gene segment of the Egyptian H5N1 divergent viruses and applied to nationwide surveillance. The modified RRT-PCR assay was more sensitive than the original one in the detection of Egyptian isolates, with 104% amplification efficiency. Sixty-one field samples were found to be positive in our assay, but only 51 samples tested positive by the original protocol and were more sensitive than matrix gene RRT-PCR detection assay. A detection limit of 10 mean embryo infective dose (EID50) with the updated oligonucleotides primers and probe set was found. For the foreseeable future, mutation of H5N1 viruses and the endemic situation in developing countries require continuous improvement of current diagnostics to aid in the containment of the H5N1 virus in poultry sectors and to lower the threat of influenza virus spread.

Isolation of highly pathogenic avian influenza H5N1 from table eggs after vaccinal break in commercial layer flock.

In May 2009, during routine monitoring of a commercial layer flock of about 87,000 birds kept in cages in 4 different houses that had been vaccinated 3 times with an inactivated H5N1 vaccine at weeks 1, 7, and 16, highly pathogenic avian influenza (HPAI) virus of subtype H5N1 was isolated and detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in tracheal and cloacal swabs collected from houses 3 and 4; 7 days after onset of clinical signs, there was an increase in mortality accompanied by a decrease in egg production and egg quality. In addition, using RT-PCR, the viral RNA could be detected from albumin and eggshell as well. Seven days after the onset of the clinical signs, the hemagglutination inhibition (HI) titers in the affected houses were 3.2 and 1.9 log2. In the other two houses, there were no clinical signs, and all tested samples were negative using virus isolation and real-time RT-PCR. The HI titers were 6.6 and 7.0 log2 in nonaffected houses. The isolated virus from egg albumin showed high nucleotides and amino-acid identities and clustered with viruses from recently H5N1-confirmed human infections and poultry from different places in Egypt. Moreover, several amino-acid substitutions of viral H5 protein were observed. The vaccinal break seems to be associated with immune escape mutants and/or improper vaccination. The role of contaminated eggs as a source of infection and as a vehicle for spread of the virus should be considered in area with avian influenza outbreaks.

Circulation of avian influenza H5N1 in live bird markets in Egypt.

The poultry meat trade in Egypt depends mainly on live bird markets (LBMs) because of insufficient slaughterhouses, lack of marketing infrastructure, and cultural preference for consumption of freshly slaughtered poultry. There are two types of LBMs in Egypt: retail shops and traditional LBMs where minimal, if any, food safety standards or veterinary inspection are implemented. Before January 2009, LBMs were considered to be a missing link in the epidemiology of avian influenza in Egypt. This incited us to initiate this surveillance to better understand the perpetuation of H5N1 and the risk of infection in poultry markets. Seventy-one out of 573 (12.4%) examined LBMs were positive for the H5N1 subtype by real-time--quantitative polymerase chain reaction (RT-qPCR) from January to April 2009. Where a 70.4% detection rate from LBMs had waterfowl only as a solitary sold species, a 26.8% detection rate from LBMs had waterfowl mixed with chicken and/or turkey, and 2.8% from LBMs had only turkey. Higher incidence, 40.8%, of positive LBMs was recorded during the cold month of February and concentrated mainly in the highly populated Nile Delta. These findings revealed wide circulation of H5N1 avian influenza virus in LBMs in Egypt, which poses a threat to public health and the poultry industry. Long-term control measures are required, and routine surveillance of bird markets should be conducted year-round.

Avian influenza H5N1 virus infections in vaccinated commercial and backyard poultry in Egypt.

In this paper, we describe results from a high-pathogenic H5N1 avian influenza virus (AIV) surveillance program in previously H5-vaccinated commercial and family-backyard poultry flocks that was conducted from 2007 to 2008 by the Egyptian National Laboratory for Veterinary Quality Control on Poultry Production. The real-time reverse transcription PCR assay was used to detect the influenza A virus matrix gene and detection of the H5 and N1 subtypes was accomplished using a commercially available kit real-time reverse transcription PCR assay. The virus was detected in 35/3,610 (0.97%) and 27/8,682 (0.31%) of examined commercial poultry farms and 246/816 (30%) and 89/1,723 (5.2%) of backyard flocks in 2007 and 2008, respectively. Positive flocks were identified throughout the year, with the highest frequencies occurring during the winter months. Anti-H5 serum antibody titers in selected commercial poultry ranged from <2 (negative) to 9.6 log(2) when determined in the hemagglutination inhibition test using a H5 AIV antigen. In conclusion, despite the nationwide vaccination strategy of poultry in Egypt to combat H5N1 AIV, continuous circulation of the virus in vaccinated commercial and backyard poultry was reported and the efficacy of the vaccination using a challenge model with the current circulating field virus should be revised.

Biological fitness and natural selection of amantadine resistant variants of avian influenza H5N1 viruses.

Outbreaks caused by the highly pathogenic H5N1 avian influenza virus (A/H5N1) devastated the poultry industry in several countries and posed a significant pandemic threat. In addition to culling of infected poultry and vaccination, amantadine has been applied in poultry in some countries to control the spread of the virus. The prevalence of the amantadine resistance marker at position 31 (Ser31Asn) of the M2 protein increased over time. However, little is known about the biological fitness and selection of H5N1 amantadine resistant strains over their sensitive counterparts. Here, using reverse genetics we investigated the biological impact of Ser31Asn in M2 commonly seen in viruses in clade 2.2.1.1 in farmed poultry in Egypt. Findings of the current study indicated that the resistance to amantadine conferred by Asn31 evolved rapidly after the application of amantadine in commercial poultry. Both the resistant and sensitive strains replicated at similar levels in avian cell culture. Asn31 increased virus entry into the cells and cell-to-cell spread and was genetically stable for several passages in cell culture. Moreover, upon co-infection of cell culture resistant strains dominated sensitive viruses even in the absence of selection by amantadine. Together, rapid emergence, stability and domination of amantadine-resistant variants over sensitive strains limit the efficacy of amantadine in poultry.