E. M. Abdelwhab

Circulation of Avian Influenza H5N1 in Live Bird Markets in Egypt

Abstract 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.

Influence of maternal immunity on vaccine efficacy and susceptibility of one day old chicks against Egyptian highly pathogenic avian influenza H5N1

In Egypt, continuous circulation of highly pathogenic avian influenza (HPAI) H5N1 viruses of clade 2.2.1 in vaccinated commercial poultry challenges strenuous control efforts. Here, vaccine-derived maternal AIV H5 specific immunity in one-day old chicks was investigated as a factor of vaccine failure in long-term blanket vaccination campaigns in broiler chickens. H5 seropositive one-day old chicks were derived from breeders repeatedly immunized with a commercial inactivated vaccine based on the Potsdam/H5N2 strain. When challenged using the antigenically related HPAIV strain Italy/98 (H5N2) clinical protection was achieved until at least 10 days post-hatch although virus replication was not fully suppressed. No protection at all was observed against the Egyptian HPAIV strain EGYvar/H5N1 representing a vaccine escape lineage. Other groups of chicks with maternal immunity were vaccinated once at 3 or 14 days of age using either the Potsdam/H5N2 vaccine or a vaccine based on EGYvar/H5N1. At day 35 of age these chicks were challenged with the Egyptian HPAIV strain EGYcls/H5N1 which co-circulates with EGYvar/H5N1 but does not represent an antigenic drift variant. The Potsdam/H5N2 vaccinated groups were not protected against EGYcls/H5N1 infection while, in contrast, the EGYvar/H5N1 vaccinated chicks withstand challenge with EGYvar/H5N1 infection. In addition, the results showed that maternal antibodies could interfere with the immune response when a homologous vaccine strain was used.

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.

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.

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

Abstract 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.

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.

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

BackgroundThe 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.MethodsHere 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.ResultsIn 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).ConclusionOur 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.

Insight into alternative approaches for control of avian influenza in poultry, with emphasis on highly pathogenic H5N1.

Highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 causes a devastating disease in poultry but when it accidentally infects humans it can cause death. Therefore, decrease the incidence of H5N1 in humans needs to focus on prevention and control of poultry infections. Conventional control strategies in poultry based on surveillance, stamping out, movement restriction and enforcement of biosecurity measures did not prevent the virus spreading, particularly in developing countries. Several challenges limit efficiency of the vaccines to prevent outbreaks of HPAIV H5N1 in endemic countries. Alternative and complementary approaches to reduce the current burden of H5N1 epidemics in poultry should be encouraged. The use of antiviral chemotherapy and natural compounds, avian-cytokines, RNA interference, genetic breeding and/or development of transgenic poultry warrant further evaluation as integrated intervention strategies for control of HPAIV H5N1 in poultry.

Evolutionary trajectories and diagnostic challenges of potentially zoonotic avian influenza viruses H5N1 and H9N2 co-circulating in Egypt.

In Egypt, since 2006, descendants of the highly pathogenic avian influenza virus (HP AIV) H5N1 of clade 2.2 continue to cause sharp losses in poultry production and seriously threaten public health. Potentially zoonotic H9N2 viruses established an endemic status in poultry in Egypt as well and co-circulate with HP AIV H5N1 rising concerns of reassortments between H9N2 and H5N1 viruses along with an increase of mixed infections of poultry. Nucleotide sequences of whole genomes of 15 different isolates (H5N1: 7; H9N2: 8), and of the hemagglutinin (HA) and neuraminidase (NA) encoding segments of nine further clinical samples (H5N1: 2; H9N2: 7) from 2013 and 2014 were generated and analysed. The HA of H5N1 viruses clustered with clade 2.2.1 while the H9 HA formed three distinguishable subgroups within cluster B viruses. BEAST analysis revealed that H9N2 viruses are likely present in Egypt since 2009. Several previously undescribed substituting mutations putatively associated with host tropism and virulence modulation were detected in different proteins of the analysed H9N2 and H5N1 viruses. Reassortment between HP AIV H5N1 and H9N2 is anticipated in Egypt, and timely detection of such events is of public health concern. As a rapid tool for detection of such reassortants discriminative SYBR-Green reverse transcription real-time PCR assays (SG-RT-qPCR), targeting the internal genes of the Egyptian H5N1 and H9N2 viruses were developed for the rapid screening of viral RNAs from both virus isolates and clinical samples. However, in accordance to Sanger sequencing, no reassortants were found by SG-RT-qPCR. Nevertheless, the complex epidemiology of avian influenza in poultry in Egypt will require sustained close observation. Further development and continuing adaptation of rapid and cost-effective screening assays such as the SG-RT-qPCR protocol developed here are at the basis of efforts for improvement the currently critical situation.

The Neuraminidase Stalk Deletion Serves as Major Virulence Determinant of H5N1 Highly Pathogenic Avian Influenza Viruses in Chicken.

Highly pathogenic avian influenza viruses (HPAIV) cause devastating losses in gallinaceous poultry world-wide and raised concerns of a novel pandemic. HPAIV develop from low-pathogenic precursors by acquisition of a polybasic HA cleavage site (HACS), the prime virulence determinant. Beside that HACS, other adaptive changes accumulate in those precursors prior to transformation into an HPAIV. Here, we aimed to unravel such virulence determinants in addition to the HA gene. Stepwise reduction of HPAIV genes revealed that the HPAIV HA and NA form a minimum set of virulence determinants, sufficient for a lethal phenotype in chicken. Abolishing the NA stalk deletion considerably reduced lethality and prevented transmission. Conversely, the analogous stalk deletion reconstructed in the NA of an LPAIV reassortant carrying only the HPAIV HA resulted in 100% lethality both after primary and contact infection. Remarkably, the unmodified LPAIV NA with its long stalk, when exclusively introduced into the H5N1 HPAIV, still enabled high virulence and efficient transmission. Therefore, irrespective of an NA stalk deletion, minor virulence determinants in addition to the essential polybasic HACS contribute to high virulence, whereas the NA stalk deletion alone may serve as major virulence determinant.