Christian Grund

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.

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.

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.

Evaluation of two commercial loop-mediated isothermal amplification assays for detection of avian influenza H5 and H7 hemagglutinin genes.

Real-time reverse transcription loop–mediated isothermal amplification (real-time RT-LAMP) holds substantial potential as a highly sensitive, specific, and easy-to-perform molecular technique for pathogen detection in clinical samples. In the current study, the analytical and diagnostic performance of 2 commercial realtime RT-LAMP kits, Avian Flu H5 and Avian Flu H7, in detecting Avian influenza virus (AIV) infections were evaluated and compared with validated real-time reverse transcription polymerase chain reaction (RT-PCR) assays using RNA from reference virus isolates of subtypes H5 (n = 24) and H7 (n = 25) and of phylogenetically related subtypes (n = 20). When real-time RT-LAMP was carried out according to the recommendations of the manufacturer, 3 out of 24 H5 isolates and 8 out of 25 H7 reference strains were not detected. Prolonging the amplification phase resulted in detection of all H5 isolates but also in false positive detection of 2 non-H5 isolates. Real-time RT-LAMP specific to H7 failed to detect 2 H7 isolates after prolonged amplification. According to the examination of RNA log dilutions, the sensitivity of the real-time RT-LAMP assays, for a number of historic but also recent strains, was considerably lower compared with subtype-specific real-time RT-PCR assays. Application of the real-time RT-LAMP assays for analysis of diagnostic samples from wild birds confirmed their lower sensitivity. Commercial real-time RT-LAMP as tested in this study with a broad range of AIV H5 and H7 strains of phylogenetically diverse yet recent origin, holds some promise for routine veterinary diagnostic purposes, although real-time RT-LAMP was markedly more vulnerable to a reduction of detection limits because of strain-specific sequence variation than subtype-specific real-time RT-PCR.

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.

Consecutive natural influenza a virus infections in sentinel mallards in the evident absence of subtype-specific hemagglutination inhibiting antibodies.

Dabbling ducks, particularly Mallards (Anas platyrhynchos) have been frequently and consistently reported to play a pivotal role as a reservoir of low pathogenic avian influenza viruses (AIV). From October 2006 to November 2008, hand-raised Mallard ducks kept at a pond in an avifaunistically rich area of Southern Germany served as sentinel birds in the AIV surveillance programme in Germany. The pond was regularly visited by several species of dabbling ducks. A flock of sentinel birds, consisting of the same 16 individual birds during the whole study period, was regularly tested virologically and serologically for AIV infections. Swab samples were screened by RT-qPCR and, if positive, virus was isolated in embryonated chicken eggs. Serum samples were tested by the use of competitive ELISA and hemagglutinin inhibition (HI) assay. Sequences of full-length hemagglutinin (HA) and neuraminidase (NA) genes were phylogenetically analysed. Four episodes of infections with Eurasian-type AIV occurred in August (H6N8), October/November (H3N2, H2N3) 2007, in January (H3N2) and September (H3N8) 2008. The HA and NA genes of the H3N2 viruses of October 2007 and January 2008 were almost identical rendering the possibility of a re-introduction of that virus from the environment of the sentinel flock highly likely. The HA of the H3N8 virus of September 2008 belonged to a different cluster. As a correlate of the humoral immune response, titres of nucleocapsid protein-specific antibodies fluctuated in correlation with the course of AIV infection episodes. However, no specific systemic response of hemagglutination inhibiting antibodies could be demonstrated even if homologous viral antigens were used. Besides being useful as early indicators for the circulation of influenza viruses in a specific region, the sentinel ducks also contributed to gaining insights into the ecobiology of AIV infection in aquatic wild birds.

Broad spectrum reactivity versus subtype specificity-Trade-offs in serodiagnosis of influenza A virus infections by competitive ELISA

Avian influenza viruses (AIVs) of the H5 and H7 subtypes can cause substantial economic losses in the poultry industry and are a potential threat to public health. Serosurveillance of poultry populations is an important monitoring tool and can also be used for control of vaccination campaigns. The purpose of this study was to develop broadly reactive, yet subtype-specific competitive ELISAs (cELISAs) for the specific detection of antibodies to the notifiable AIV subtypes H5 and H7 as an alternative to the gold standard haemagglutination inhibition assay (HI). Broadly reacting monoclonal competitor antibodies (mAbs) and genetically engineered subtype H5 or H7 haemagglutinin antigen, expressed and in vivo biotinylated in insect cells, were used to develop the cELISAs. Sera from galliform species and water fowl (n=793) were used to evaluate the performance characteristics of the cELISAs. For the H5 specific cELISA, 98.1% test sensitivity and 91.5% test specificity (97.7% and 90.2% for galliforms; 98.9% and 92.6% for waterfowl), and for the H7 cELISA 97.3% sensitivity and 91.8% specificity (95.3% and 98.9% for galliforms; 100% and 82.7% for waterfowl) were reached when compared to HI. The use of competitor mAbs with broad spectrum reactivity within an AIV haemagglutinin subtype allowed for homogenous detection with high sensitivity of subtype-specific antibodies induced by antigenically widely distinct isolates including antigenic drift variants. However, a trade-off regarding sensitivity versus nonspecific detection of interfering antibodies induced by phylo- and antigenically closely related subtypes, e.g., H5 versus H2 and H7 versus H15, must be considered. The observed intersubtype antibody cross-reactivity remains a disturbance variable in AIV subtype-specific serodiagnosis which negatively affects specificity.

In vivo biotinylated recombinant influenza A virus hemagglutinin for use in subtype-specific serodiagnostic assays.

There is an urgent need for robust subtype-specific serological tests to diagnose influenza A virus infections in poultry and mammals, including humans. Such assays require reliable subtype-specific sources of soluble and authentically folded seroreactive hemagglutinin (HA), one of the integral membrane proteins that determine the serological subtype of influenza viruses. To this purpose, a bigenic pFastBacDual baculovirus transfer vector allowing efficient invivo biotinylation of soluble HA homo-oligomers expressed via the secretory pathway was developed. An Avi-Tag allowed site-specific biotinylation by a coexpressed genetically modified BirA biotin ligase retained in the endoplasmic reticulum (ER). Highly seroreactive mono-biotinylated HA of recent H5 and H7 influenza A subtypes was secreted from recombinant baculovirus infected High-Five insect cells at levels sufficient to directly load streptavidin-coated enzyme-linked immunosorbent assay (ELISA) matrices, thereby avoiding any purification steps. The recombinant antigens retained authentic antigenicity, including conformation-dependent epitopes involved in hemagglutination inhibition as detected by monoclonal antibodies. This is the first bigenic invivo biotinylation system established for use in insect cells with secretable recombinant membrane proteins biotinylated by an ER-retained variant of BirA biotin ligase. The proposed technique is expected to significantly increase flexibility in the design of subtype-specific assays, thereby expanding the power of influenzaA virus serodiagnosis.