Retno D. Soejoedono

A single vaccination of commercial broilers does not reduce transmission of H5N1 highly pathogenic avian influenza

Vaccination of chickens has become routine practice in Asian countries in which H5N1 highly pathogenic avian influenza (HPAI) is endemically present. This mainly applies to layer and breeder flocks, but broilers are usually left unvaccinated. Here we investigate whether vaccination is able to reduce HPAI H5N1 virus transmission among broiler chickens. Four sets of experiments were carried out, each consisting of 22 replicate trials containing a pair of birds. Experiments 1-3 were carried out with four-week-old birds that were unvaccinated, and vaccinated at day 1 or at day 10 of age. Experiment 4 was carried out with unvaccinated day-old broiler chicks. One chicken in each trial was inoculated with H5N1 HPAI virus. One chicken in each trial was inoculated with virus. The course of the infection chain was monitored by serological analysis, and by virus isolation performed on tracheal and cloacal swabs. The analyses were based on a stochastic SEIR model using a Bayesian inferential framework. When inoculation was carried out at the 28th day of life, transmission was efficient in unvaccinated birds, and in birds vaccinated at first or tenth day of life. In these experiments estimates of the latent period (~1.0 day), infectious period (~3.3 days), and transmission rate parameter (~1.4 per day) were similar, as were estimates of the reproduction number (~4) and generation interval (~1.4 day). Transmission was significantly less efficient in unvaccinated chickens when inoculation was carried out on the first day of life. These results show that vaccination of broiler chickens does not reduce transmission, and suggest that this may be due to the interference of maternal immunity.

Efficacy of a Recombinant HVT-H5 Vaccine Against Challenge with Two Genetically Divergent Indonesian HPAI H5N1 Strains

SUMMARY. The swift evolution rate of avian influenza (AI) H5N1 virus demands constant efforts to update inactivated vaccines to match antigenically with the emerging new field virus strains. Recently, a recombinant turkey herpesvirus (rHVT)-AI vaccine, rHVT-H5, expressing the HA gene of a highly pathogenic avian influenza (HPAI) H5N1 clade 2.2 A/Swan/Hungary/499/2006 strain inserted into FC-126 strain of HVT vector, has been developed to combat current threats in poultry industry. Here, we present the results of two trials where rHVT-H5 was tested alone or in combination with inactivated H5N1 vaccines (the latter vaccines contained antigens produced by using a clade 2.1.3 HPAI H5N1 virus [A/Ck/WestJava-Nagrak/2007] in the first trial or mixture of antigen produced by strain A/Ck/WestJava-Nagrak/2007 and A/Ck/Banten-Tangerang/2010 [bivalent vaccine] for second trial) in broiler chickens (Gallus gallus domesticus) carrying maternally derived antibodies to H5N1 and then challenged with Indonesian HPAI H5N1 field isolates. The effectiveness of vaccination was evaluated on the basis of clinical protection (morbidity and mortality) and measurement of virus shedding after challenge. Immune response to vaccination was followed by serology. In the first experiment, chickens were vaccinated at the day of hatch with rHVT-H5 alone (Group 1) or combined with inactivated vaccine at day old (Group 2) or at 10 days of age (Group 3). The chickens along with nonvaccinated hatch-mates were challenged at 28 days of age with the HPAI H5N1 field isolate clade 2.1.3 A/Chicken/WestJava-Subang/29/2007. Eighty, 100%, and 80% clinical protection was recorded in Group 1, 2, and 3, respectively. A similar experiment was performed a second time, but the chicks in Group 3 received the inactivated vaccine earlier, at 7 days of age. Challenge was performed at 28 days of age using a different H5N1 isolate, clade 2.1.3 A/Ck/Purwakarta-Cilingga/142/10. Clinical protection achieved in the second trial was 95%, 75%, and 90% in Group 1, 2, and 3, respectively. Shedding of challenge virus was significantly lower in the vaccinated groups compared with controls in both experiments. Vaccinated birds developed hemagglutination inhibition antibody response to H5N1 by the time of challenge. These experiments confirmed that the rHVT-H5 vaccine applied alone or in combination with inactivated H5N1 vaccines could provide high level (>80%) clinical protection against divergent HPAI H5N1 field isolates after single immunization by 4 wk of age and a significant reduction in the excretion of challenge virus.

Complementary monoclonal antibody-based dot ELISA for universal detection of H5 avian influenza virus.

BackgroundRapid diagnosis and surveillance for H5 subtype viruses are critical for the control of H5N1 infection.ResultsIn this study, H5 Dot ELISA, a rapid test for the detection of avian H5N1 influenza virus, was developed with two complementary H5 monoclonal antibodies. HA sequencing of escape mutants followed by epitope mapping revealed that the two Mabs target the epitope component (189th amino acid) on the HA protein but are specific for different amino acids (189Lys or 189Arg). Gene alignment indicated that these two amino acids are the most frequent types on this position among all of the H5 AIV reported in GeneBank. These two H5 Mabs were used together in a dot ELISA to detect H5 viral antigen. The detection limit of the developed test for multiple clades of H5N1 viruses, including clades 0, 1, 2.1, 2.2, 2.3, 4, 7, and 8, was less than 0.5 hemagglutinin units. The specificity of the optimized dot ELISA was examined by using 100 H5 strains, including H5N1 HPAI strains from multiple clades, 36 non-H5N1 viruses, and 4 influenza B viruses. No cross-reactivity was observed for any of the non-H5N1 viruses tested. Among 200 random poultry samples, the test gave 100% positive results for all of the twelve RT-PCR-positive samples.ConclusionsConsidering that the test is convenient for field use, this H5 Dot ELISA can be used for on-site detection of H5N1 infection in clinical or environmental specimens and facilitate the investigation of H5N1 influenza outbreaks and surveillance in poultry.

The large-scale production of an artificial influenza virus-like particle vaccine in silkworm pupae.

We successfully established a mass production system for an influenza virus-like particle (VLP) vaccine using a synthetic H5 hemagglutinin (HA) gene codon-optimized for the silkworm. A recombinant baculovirus containing the synthetic gene was inoculated into silkworm pupae. Four days after inoculation, the hemagglutination titer in homogenates from infected pupae reached a mean value of 0.8 million hemagglutination units (HAU), approximately 2,000 μg HA protein per pupa, more than 50-fold higher than that produced with an embryonated chicken egg. VLPs ranging from 30 nm to 300 nm in diameter and covered with a large number of spikes were detected in the homogenates. The spikes were approximately 14 nm long, similar to an authentic influenza HA spike. Detailed electron micrographs indicated that the VLP spike density was similar to that of authentic influenza virus particles. The results clearly show that the expression of a single HA gene can efficiently produce VLPs in silkworm pupae. When chickens were immunized with the pupae homogenate, the hemagglutination inhibition titer in their sera reached values of 2,048-8,192 after approximately 1 month. This is the first report demonstrating that a large amount of VLP vaccine could be produced by single synthetic HA gene in silkworm pupae. Our system might be useful for future vaccine development against other viral diseases.

Experimental and Field Results Regarding Immunity Induced by a Recombinant Turkey Herpesvirus H5 Vector Vaccine Against H5N1 and Other H5 Highly Pathogenic Avian Influenza Virus Challenges

SUMMARY: Vaccination against H5N1 highly pathogenic avian influenza (AI) virus (HPAIV) is one of the possible complementary means available for affected countries to control AI when the disease has become, or with a high risk of becoming, endemic. Efficacy of the vaccination against AI relies essentially, but not exclusively, on the capacity of the vaccine to induce immunity against the targeted virus (which is prone to undergo antigenic variations), as well as its capacity to overcome interference with maternal immunity transmitted by immunized breeding hens to their progeny. This property of the vaccine is a prerequisite for its administration at the hatchery, which assures higher and more reliable vaccine coverage of the populations than vaccination at the farm. A recombinant vector vaccine (Vectormune® AI), based on turkey herpesvirus expressing the hemagglutinin gene of an H5N1 HPAIV as an insert, has been used in several experiments conducted in different research laboratories, as well as in controlled field trials. The results have demonstrated a high degree of homologous and cross protection against different genetic clades of the H5N1 HPAIV. Furthermore, vaccine-induced immunity was not impaired by the presence of passive immunity, but on the contrary, cumulated with it for improved early protection. The demonstrated levels of protection against the different challenge viruses exhibited variations in terms of postchallenge mortality, as well as challenge virus shedding. The data presented here highlight the advantages of this vaccine as a useful and reliable tool to complement biosecurity and sanitary policies for better controlling the disease due to HPAIV of H5 subtypes, when the vaccination is applied as a control measure.

Potential risk of viral transmission from flying foxes to domestic animals and humans on the southern coast of West Java, Indonesia

Flying foxes have been considered to be involved in the transmission of serious infectious diseases to humans. Using questionnaires, we aimed to determine the direct and/or indirect contacts of flying foxes in an Indonesian nature conservation area with domestic animals and humans living in the surrounding area. We surveyed 150 residents of 10 villages in West Java. Villages were classified into 3 groups: inside and/or within 1 km from the outer border of the conservation area and 1–5 km or 5–10 km away from the reserve’s outer border. Data were collected by direct interview using a structured questionnaire consisting of the respondent characteristics (age, sex and occupation); histories of contacts between flying foxes and humans, dogs and other domestic animals; and knowledge about infectious diseases, mainly rabies, in flying foxes. We found that flying foxes from the nature conservation area often enter residential areas at night to look for food, especially during the fruit season. In these residential areas, flying foxes had direct contacts with humans and a few contacts with domestic animals, especially dogs. People who encounter flying foxes seldom used personal protective equipment, such as leather gloves, goggles and caps. The residents living around the conservation area mostly had poor knowledge about flying foxes and disease transmission. This situation shows that the population in this region is at a quite high risk for contracting infectious diseases from flying foxes.

Identification of Pathogenecity of Avian Influenza Virus Subtype H5N1 from Waterfowls Base on Amino Acid Sequence of Cleavage Site Hemagglutinin Protein

Identification of pathotype of Avian Influenza Virus (AIV) subtype H5N1 isolates is very important. This research aimed to identify the pathotype of AIV subtype H5N1 isolated from household waterfowls in West Java based on molecular markers of amino acid sequences of the Hemagglutinin (HA) cleavage site. Fragments of HA genes of 21 isolates were amplified using RT-PCR with a primer pair that flanking the cleavage site region, and sequenced with dideoxy-termination method with ABI automatic sequencer (Applied Biosystems). Multiple alignment of nucleotide and their deduced amino acid sequence were analyzed using ClustalW from MEGA 3.1 program. The result shows that all H5N1 isolates (21 isolates) possess polybasic cleavage sites with 2 patterns of amino acid sequence, i.e QRERRRKKR (20 isolates) and QRESRRKKR (1 isolate). This finding indicates that all of the viruses isolated in this research were of highly pathogenic avian influenza (HPAI) strains. Keywords: cleavage site, waterfowls, HPAI

Molecular analysis of Hemagglutinin Gen of Highly Pathogenic Avian Influenza of H5N1 Subtype Isolated from Waterfowls

Avian influenza viruses (AIV) subtype H5N1 isolated from waterfowls in West Java pose the known characteristic of highly pathogenic strains, with polybasic amino acid sequence of cleavage site QRERRRKKR and QRESRRKKR. This research aimed to analyze the important domains of hemagglutinin (HA) gene of those isolates. Fragment of HA gene was amplified using RT-PCR method with specifically-designed primer pairs and sequenced using dideoxy termination method with ABI automatic sequencer (Applied Biosystems). Multiple alignment of nucleotide and deduced amino acid sequences were analyzed using ClustalW of MEGA-3.1 program. Some of biological domains of HA, i.e. antigenic sites, receptor binding pocket, and glycosylation sites of the isolates were polymorphic. The viruses also pose conserved glutamine (Q) and glisin (G) residues at the known receptor binding site, at the position 222 and 224 respectively. This findings clearly show that all AIV subtype H5N1 isolaed from waterfowl preservesthe α-2,3NeuAcGal avian receptor specificity. Key Words : Antigenic Sites, Glycosylation Sites, Receptor Binding Pocket, AIV H5N1, Waterfowls