El-Sayed M. Abdelwhab

Anatomical distribution of avian bornavirus in parrots, its occurrence in clinically healthy birds and ABV-antibody detection

Proventricular dilatation disease (PDD) is a fatal infectious disease of birds that primarily affects psittacine birds. Although a causative agent has not been formally demonstrated, the leading candidate is a novel avian bornavirus (ABV) detected in post-mortem tissue samples of psittacids with PDD from the USA, Israel and, recently, Germany. Here we describe the presence of ABV in a parrot with PDD as well as in clinically normal birds exposed to birds with PDD. In two ABV-positive post-mortem cases, the tissue distribution of ABV was investigated by quantitative real-time reverse transcription-polymerase chain reaction. Viraemia was observed in a PDD-affected bird whereas a restriction of ABV to nerve tissue was found in the non-PDD-affected bird. Healthy birds from the same aviary as the affected birds were also found to harbour the virus; 19/59 (32.2%) birds tested positive for ABV RNA in cloacal swabs, providing the first evidence of ABV in clinically healthy birds. In contrast, 39 birds from the same geographic area, but from two different aviaries without PDD cases in recent years, had negative cloacal swabs. ABV RNA-positive, clinically healthy birds demonstrated the same serological response as the animal with confirmed PDD. These results indicate that ABV infection may occur without clinical evidence of PDD and suggest that cloacal swabs can enable the non-invasive detection of ABV infection.

Multiple dose vaccination with heterologous H5N2 vaccine: immune response and protection against variant clade 2.2.1 highly pathogenic avian influenza H5N1 in broiler breeder chickens.

Circulation of an antigenically variant lineage of highly pathogenic avian influenza (HPAI) H5N1 virus in chicken breeder flocks in Egypt is a continuing problem. The protective efficacy of multiple repeated vaccinations using the currently available H5N2 vaccines is unclear. Here, broiler breeder chickens were vaccinated at weeks 6, 12 and 18 with an inactivated H5N2 commercial vaccine. HI-titer against an Egyptian H5N1 field isolate of classic clade 2.2.1 (EGYcls/H5N1) were significantly lower after the first immunization but increased after booster vaccinations. In contrast, no HI titers were induced against an antigenically distinct field virus of the variant lineage of clade 2.2.1 (EGYvar/H5N1). Upon challenge at week 50 mild, if any, clinical signs were observed in the group infected with EGYcls/H5N1 although one of eight (12.5%) birds died. Mortality reached 6/8 (75%) in the EGYvar/H5N1 challenge group. Virus excretion in all vaccinated groups was reduced in amplitude, but in vaccinated surviving birds, time of virus excretion was extended to up to ten days. Strikingly, challenged vaccinated birds kept laying eggs almost throughout the observation period. Virus was detected on the outer egg-shell of 17 of 40 eggs. The majority of the infected eggs were derived from the EGYcls/H5N1 challenged animals; here the virus was detected also in the yolk and albumin. Repeated vaccination using a commercial H5N2-based vaccine broadened the antigen profile of induced antibodies but did not provide adequate protection against heterologous virus variant. In addition, the observation of AIV contaminated eggs from infected flocks highlights the risk of silent virus spread by vaccinated animals and point to eggs as a possible vector.

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

BackgroundUninterrupted 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.FindingsIn 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.ConclusionsDespite 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.

Increasing Prevalence of Unique Mutation Patterns in H5N1 Avian Influenza Virus HA and NA Glycoproteins from Human Infections in Egypt

Highly pathogenic avian influenza H5N1 virus (HPAIV) continues to be a candidate of a further influenza virus pandemic. Egypt is the country worst affected by human cases of HPAIV H5N1 infection in 2009. Increased infection of preschool children and decreased mortality rates suggested subtle changes in the epidemiology of the infection. Among other factors, the evolution of several conspicuous viral genetic markers in the HA and NA genes of HPAIV H5N1 viruses of human cases from Egypt and their putative influence on biological virus characteristics described here may contribute to this situation.

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

BackgroundThe 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 (2.2.1v) appears to be perpetuated mainly in commercial poultry farms in Egypt. Viruses of the 2.2.1v 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.ResultsWe 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.ConclusionsThe 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.

Modified H5 Real-Time Reverse Transcriptase–PCR Oligonucleotides for Detection of Divergent Avian Influenza H5N1 Viruses in Egypt

Abstract 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.

A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens

ABSTRACT In 1999, after circulation for a few months in poultry in Italy, low-pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS), PEIPKGSRVRR*GLF (asterisk indicates the cleavage site), in the hemagglutinin (HA) and additional alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 virus (Lp) and HPAI A/chicken/Italy/445/1999 virus (Hp) were generated. Hp with a monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens, thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp_CS445) was less virulent than Hp, full virulence was exhibited when HA2 was replaced by that of Hp. In HA2, three amino acid differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp_CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental to replication in cell culture, probably due to low-pH activation of HA. A436T and/or K536R restored viral replication in vitro and in vivo. Viruses possessing A436T and K536R were observed early in the HPAI outbreak but were later superseded by viruses carrying all three mutations. Together, besides the mCS, stepwise mutations in HA2 increased the fitness of the Italian H7N1 virus in vivo. The shift toward higher virulence in the field was most likely gradual with rapid optimization. IMPORTANCE In 1999, after 9 months of circulation of low-pathogenic (LP) avian influenza virus (AIV), a devastating highly pathogenic (HP) H7N1 AIV emerged in poultry, marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main finding in this report is the identification of three mutations in the HA2 domain that are required for replication and stability, as well as for virulence, transmission, and tropism of H7N1 in chickens. In addition to the mCS, Q450L was required for full virulence and transmissibility of the virus. Nonetheless, it was detrimental to virus replication and required A436T and/or K536R to restore replication, systemic spread, and stability. These results are important for better understanding of the evolution of highly pathogenic avian influenza viruses from low-pathogenic precursors.

Molecular diagnosis and characterization of Cryptosporidium spp. in turkeys and chickens in Germany reveals evidence for previously undetected parasite species

A total of 256 fecal specimens were randomly collected from farmed poultry in Germany and screened for the presence of Cryptosporidium spp. by PCR and further characterized by direct automated DNA sequencing. Using a nested PCR amplifying approximately 830 bp 18S rDNA fragment, 7.03% (n = 18) of the samples were Cryptosporidium-positive. In detail, Cryptosporidium was detected in 9.3% (8/86) of turkeys, 5.7% (9/158) of broilers and 8.3% (1/12) of layers. After DNA sequencing, Cryptosporidium parvum the most frequently observed species was identified in 5.1% (13/256) of all poultry species, including 8.1% (7/86) of turkeys, 3.2% (5/158) of broilers and 8.3% (1/12) of layers. Cryptosporidium baileyi was detected in 1.3% (2/256) of the broilers only. Three novel unclassified Cryptosporidium spp. were detected in 1.2% (1/86) of turkeys and 1.3% (2/158) of broilers. The infection rate was high in 13–20 week old turkeys, 1–6 weeks old broilers and >20 weeks old layers but differences between age groups were not significant. This is the first study in Germany uses molecular methods for the detection of Cryptosporidium in poultry. The results indicate that Cryptosporidium parasites are common among broilers and turkeys in Germany. Considering the large size of the poultry industry, the large amount of poultry meat that is consumed and the fact that C. parvum is also the most common Cryptosporidium parasite in humans, poultry might also be a source of human infections.

Prevalence of the C-terminal truncations of NS1 in avian influenza A viruses and effect on virulence and replication of a highly pathogenic H7N1 virus in chickens

abstract Highly pathogenic (HP) avian influenza viruses (AIV) evolve from low pathogenic (LP) precursors after circulation in poultry by reassortment and/or single mutations in different gene segments including that encoding NS1. The carboxyl terminal end (CTE) of NS1 exhibits deletions between amino acid 202 and 230 with still unknown impact on virulence of AIV in chickens. In this study, NS1 protein sequences of all AIV subtypes in birds from 1902 to 2015 were analyzed to study the prevalence and distribution of CTE truncation (ΔCTE). Thirteen different ΔCTE forms were observed in NS1 proteins from 11 HA and 8 NA subtypes with high prevalences in H9, H7, H6 and H10 and N9, N2, N6 and N1 subtypes particularly in chickens and minor poultry species. With 88% NS217 lacking amino acids 218–230 was the most common ΔCTE form followed by NS224 (3.6%). NS217 was found in 10 and 8 different HA and NA subtypes, respectively, whereas NS224 was detected exclusively in the Italian HPAIV H7N1 suggesting relevance for virulence. To test this assumption, 3 recombinant HPAIV H7N1 were constructed carrying wild-type HP NS1 (Hp-NS224), NS1 with extended CTE (Hp-NS230) or NS1 from LPAIV H7N1 (Hp-NSLp), and tested in-vitro and in-vivo. Extension of CTE in Hp NS1 significantly decreased virus replication in chicken embryo kidney cells. Truncation in the NS1 decreased the tropism of Hp-NS224 to the endothelium, central nervous system and respiratory tract epithelium without significant difference in virulence in chickens. This study described the variable forms of ΔCTE in NS1 and indicated that CTE is not an essential virulence determinant particularly for the Italian HPAIV H7N1 but may be a host-adaptation marker required for efficient virus replication.

Progressive glycosylation of the haemagglutinin of avian influenza H5N1 modulates virus replication, virulence and chicken-to-chicken transmission without significant impact on antigenic drift

Highly pathogenic H5N1 avian influenza virus (A/H5N1) devastated the poultry industry and continues to pose a pandemic threat. Studying the progressive genetic changes in A/H5N1 after long-term circulation in poultry may help us to better understand A/H5N1 biology in birds. A/H5N1 clade 2.2.1.1 antigenic drift viruses have been isolated from vaccinated commercial poultry in Egypt. They exhibit a peculiar stepwise accumulation of glycosylation sites (GS) in the haemagglutinin (HA) with viruses carrying, beyond the conserved 5 GS, additional GS at amino acid residues 72, 154, 236 and 273 resulting in 6, 7, 8 or 9 GS in the HA. Available information about the impact of glycosylation on virus fitness and pathobiology is mostly derived from mammalian models. Here, we generated recombinant viruses imitating the progressive acquisition of GS in HA and investigated their biological relevance in vitro and in vivo. Our in vitro results indicated that the accumulation of GS correlated with increased glycosylation, increased virus replication, neuraminidase activity, cell-to-cell spread and thermostability, however, strikingly, without significant impact on virus escape from neutralizing antibodies. In vivo, glycosylation modulated virus virulence, tissue tropism, replication and chicken-to-chicken transmission. Predominance in the field was towards viruses with hyperglycosylated HA. Together, progressive glycosylation of the HA may foster persistence of A/H5N1 by increasing replication, stability and bird-to-bird transmission without significant impact on antigenic drift.