Evaluating methods for Avian avulavirus-1 whole genome sequencing.

BACKGROUND: Avian avulavirus-1 (AAvV-1, previously Newcastle Disease Virus) is responsible for poultry and wild birds' disease outbreaks. Numerous whole genome sequencing methods were reported for this virus. These methods included cloning, specific primers amplification, shotgun PCR approaches, Sequence Independent Single Primer Amplification and next generation sequencing platform kits. METHODS: Three methods were used to sequence 173 Israeli Avian avulavirus-1 field isolates and one vaccine strain (VH). The sequencing was performed on Proton and Ion Torrent Personal Genome Machine and to a lesser extent, Illumina MiSeq and NextSeq sequencers. Target specific primers (SP) and Sequence Independent Single Primer Amplification (SISPA) products sequenced via the Ion torrent sequencer had a high error rate and truncated genomes. All the next generation sequencing platform sequencing kits generated high sequence accuracy and near-complete genomic size. RESULTS: A high level of mutations was observed in the intergenic regions between the avian avulavirus-1 genes. Within genes, multiple regions are more mutated than the Fusion region currently used for typing. CONCLUSIONS: Our findings suggest that the whole genome sequencing by the Ion torrent sequencing kit is sufficient. However, when higher fidelity is desired, the Illumina NextSeq and Proton torrent sequencing kits were found to be preferable.

Evaluating methods for Avian avulavirus-1 whole genome sequencing.

BACKGROUND: Avian avulavirus-1 (AAvV-1, previously Newcastle Disease Virus) is responsible for poultry and wild birds' disease outbreaks. Numerous whole genome sequencing methods were reported for this virus. These methods included cloning, specific primers amplification, shotgun PCR approaches, Sequence Independent Single Primer Amplification and next generation sequencing platform kits. METHODS: Three methods were used to sequence 173 Israeli Avian avulavirus-1 field isolates and one vaccine strain (VH). The sequencing was performed on Proton and Ion Torrent Personal Genome Machine and to a lesser extent, Illumina MiSeq and NextSeq sequencers. Target specific primers (SP) and Sequence Independent Single Primer Amplification (SISPA) products sequenced via the Ion torrent sequencer had a high error rate and truncated genomes. All the next generation sequencing platform sequencing kits generated high sequence accuracy and near-complete genomic size. RESULTS: A high level of mutations was observed in the intergenic regions between the avian avulavirus-1 genes. Within genes, multiple regions are more mutated than the Fusion region currently used for typing. CONCLUSIONS: Our findings suggest that the whole genome sequencing by the Ion torrent sequencing kit is sufficient. However, when higher fidelity is desired, the Illumina NextSeq and Proton torrent sequencing kits were found to be preferable.

Genetic and antigenic characterization of sigma C protein from avian reovirus.

Avian reovirus (ARV) causes viral arthritis, tenosynovitis, liver infection and immunosuppression in birds. Live-attenuated and inactivated vaccines for ARV are available, but do not efficiently protect against recent variants. Sigma C, which mediates virus attachment to target cells, is the most variable protein in ARV. Antibodies to this protein neutralize viral infection. The purpose of the present study was to characterize sigma C in isolates of ARV from infected birds, as compared with the vaccine strain. Amino acids 27 to 293 of sigma C from 28 Israeli isolates were compared, classified and analysed using bioinformatics tools. Large variations were found among the isolates, and the vaccine strain was shown to differ from most of the studied strains, which could explain the failure of commonly used vaccinations in protecting birds against ARV infection. Based on sigma C protein sequences from all over the world, ARV can be divided into four groups. Isolates from all groups were found in the field simultaneously, possibly explaining the insufficient protection achieved by the vaccine strain, which is represented in one of the groups. The results point out the need and the difficulty in producing a wide-ranging vaccine. Several conserved regions among all reported ARV sigma C proteins were identified. These peptides were further studied for structural and functional properties, and for antigenic characterization. The results of this study shed light on peptide selection for a broad and efficient vaccine.

Phylogenetic analysis of hemagglutinin, neuraminidase, and nucleoprotein genes of H9N2 avian influenza viruses isolated in Israel during the 2000-2005 epizootic.

The first two isolates of H9N2 influenza virus in Israel were collected from turkey and chicken hosts in May 2000. The actual epizootic of the H9N2 virus started in December 2001, after a 1.5-year period of silence, and still continues. A total of more than 500 isolations from turkeys and chickens were registered during the outbreaks. The present study has revealed some genetic peculiarities among the local isolates, namely: all the isolates belong to the same G1-like phylogenetic lineage, within which they form a single group, which, in turn, is divided into three subgroups in the cases of the HA and NP genes, and two subgroups in the case of the NA gene. The results present a basis for suggesting the existence of two parallel evolutionary trends originating from the same local "prototype" isolate.

Genetic characterization of avian influenza viruses isolated in Israel during 2000-2006.

Our aim was to establish the phylogenetic and genetic relationships among avian influenza viruses (AIV) recently isolated from poultry in Israel. During this study we analyzed complete nucleotide sequences of two envelope (hemagglutinin and neuraminidase) and six internal genes (polymerase B1, polymerase B2, polymerase A, nucleoprotein, nonstructural, and matrix) of 29 selected H9N2 and six internal genes of five H5N1 viruses isolated in Israel during 2000-2006. Comparative genetic and phylogenetic analyses of these sequences revealed that the local H5N1 viruses are closely related to H5N1 viruses isolated in European, Asian, and Middle Eastern countries in 2005-2006. The H9N2 Israeli isolates, together with viruses isolated in Jordan and Saudi Arabia formed a single group. Our data support the claim that during recent years a new endemic focus of H9N2 has been formed in the Middle East. The introduction of H5N1 and co-circulation of these two subtypes of AIV in this region may augment the risk of potentially pandemic strains emergence.

Molecular characterization of the glycoprotein genes of H5N1 influenza A viruses isolated in Israel and the Gaza Strip during 2006 outbreaks.

Highly pathogenic H5N1 avian influenza A viruses (AIV) have caused outbreaks among domestic poultry and wild aquatic birds in many Asian, European, and African countries since 1997. In March 2006 an avian H5N1 influenza A virus was isolated from poultry in Israel. In the present study we molecularly characterized the hemagglutinin (HA) and neuraminidase (NA) genes of eleven H5N1 viruses isolated from domestic poultry in Israel and Gaza in March-April 2006. Phylogenetic analysis of the HA and NA genes showed that the Israeli and Gazian viruses were closely related to viruses isolated in Egypt in 2006.

Genetic analysis of nonstructural genes (NS1 and NS2) of H9N2 and H5N1 viruses recently isolated in Israel.

The avian influenza virus subtype H9N2 affects wild birds, domestic poultry, swine, and humans; it has circulated amongst domestic poultry in Israel during the last 6 years. The H5N1 virus was recorded in Israel for the first time in March 2006. Nonstructural (NS) genes and NS proteins are important in the life cycle of the avian influenza viruses. In the present study, NS genes of 21 examples of H9N2 and of two examples of H5N1 avian influenza viruses, isolated in Israel during 2000-2006, were completely sequenced and phylogenetically analyzed. All the H9N2 isolates fell into a single group that, in turn, was subdivided into three subgroups in accordance with the time of isolation; their NS1 and NS2 proteins possessed 230 and 121 amino acids, respectively. The NS1 protein of the H5N1 isolates had five amino acid deletions, which was typical of highly pathogenic H5N1 viruses isolated in various countries during 2005-2006. Comparative analysis showed that the NS proteins of the H9N2 Israeli isolates contained few amino acid sequences associated with high pathogenicity or human host specificity.

Multifocal avian influenza (H5N1) outbreak.

During March 2006, an outbreak of highly pathogenic avian influenza (H5N1) occurred in multiple poultry farms in Israel. The epidemiologic investigation and review of outbreak mitigation efforts uncovered gaps in planning for and containing the outbreak, thus affording valuable lessons applicable to other countries in similar settings.

Genetic characterization of the H9N2 influenza viruses circulated in the poultry population in Israel.

The partial nucleotide sequences of the hemagglutinin (HA) genes of 72 H9N2 influenza viruses isolated from chickens and turkeys in Israel during the period 2000-2005 were genetically analyzed. The isolates possessed the three types of amino acid motif -R-S-S-R/G-L-, -R-S-N-R/G-L-, and -R-S-K-R/G-L- at the cleavage site of HA. Phylogenetic analyses showed that all Israeli isolates belonged to the same group which further divided into three closely related sub-groups. The HA genes of these isolates were related to the HA gene of A/chicken/Germany/R45/98 isolated from chicken in Germany in 1998.