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.

Practical aspects in the use of passive immunization as an alternative to attenuated viral vaccines.

Passive immunization as a method to protect birds has been tested for many years and shown to be effective. Its advantages over active vaccination include no use of partially virulent viruses, overcoming the gap in the level of protection at young age due to interference of maternal antibodies to raise self-immune response following active vaccination and the possible immunosuppressive effect of attenuated vaccine viruses. However, a major obstacle to its implementation is its relatively high cost which is dependent, among other things, mainly on two factors: the efficacy of antibody production, and the use of specific pathogen-free (SPF) birds for antibody production to avoid the possible transfer of pathogens from commercial layers. In this study we show efficient production of immunoglobulin Y (IgY) against four different pathogens simultaneously in the same egg, and treatment of the extracted IgY with formalin to negate the need for SPF birds. Formalin, a common registered sterilization compound in vaccine production, was shown not to interfere with the Fab specific antigen binding or Fc-complement activation of the antibody. Following injection of 1-day-old broilers with antibodies against infectious bursal disease virus, protective antibody levels were acquired for the entire period of sensitivity to this pathogen (35 days). Passive vaccination with formalin-sterilized IgY against multiple antigens extracted from one commercial egg may be a cost-effective and advantageous complementary or alternative to attenuated vaccines in poultry.

Longitudinal survey of avian metapneumoviruses in poultry in Israel: infiltration of field strains into vaccinated flocks.

Avian metapneumovirus (aMPV) is a respiratory pathogen and includes four subtypes, which are differentially distributed worldwide. In Israel, two aMPV subtypes, A and B, are cocirculating in an area with a relatively high density of poultry houses. We performed a longitudinal survey in commercial flocks of turkeys and chickens (six and two flocks, respectively) with differing vaccination programs. This study revealed that all monitored flocks were infected by aMPV during the sampling period, as detected either by serology or by molecular methods. Moreover, to identify the subtypes infecting the birds and to distinguish between field and vaccine strains, we sequenced fragments of the attachment glycoprotein gene. This analysis revealed that both vaccine and field strains of both aMPV-A and -B could be recovered from the birds. In four out of six turkey flocks, aMPV field strains were recovered, emphasizing the broad distribution of this highly contagious pathogen. Importantly, in two out of four vaccinated turkey flocks, homologous field subtypes of aMPV-A or -B were detected even after boost administration, suggesting failure of the vaccine to protect the flocks from secondary infection. Overall, the results of this longitudinal study call for a more efficient vaccination program against aMPV, which is critical for an area of intensive commercial poultry farming.

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.

Experimental infection of common garter snakes (Thamnophis sirtalis) with West Nile virus.

The role of various reptilian species in the infectious cycle of several arboviruses is documented, but their role in that of West Nile virus (WNV) is uncertain. Common garter snakes (Thamnophis sirtalis) were infected subcutaneously with 10(5) plaque forming units (PFU) WNV-Isr 98, five of nine snakes became viremic, and five exhibited persistent low levels of neutralizing antibodies. Four of the parentally infected snakes died and high titers of virus were found in multiple organ samples. In contrast, orally infected garter snakes did not become viremic, but viral RNA was detected in cloacal swabs. Since oral infection of predator birds by WNV is known, their ingestion of infected snakes may also result in their becoming infected.

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.

Characterization of Israeli avian metapneumovirus strains in turkeys and chickens.

Although the disease associated with the avian metapneumovirus (aMPV) has been described in Israel for more than 20 years and one Israeli isolate was characterized as subtype A, this is the first study investigating the strains circulating in Israel (period 2002 to 2004). Viral RNA was detected by reverse transcription-polymerase chain reaction in 44% of the examined flocks, and one-half of the virus isolation attempts were successful. Characterization showed that 34 non-vaccinated turkey flocks had aMPV subtype B, three had aMPV subtype A and three remained undetermined. Sequence analysis of part of the attachment protein G gene (1161 base pairs) showed a relative homogeneity within the Israeli subtype B group, although they were different from the European and vaccine strains. Seven out of 10 vaccinated flocks were reverse transcription-polymerase chain reaction-positive for aMPV, and sequence analysis of 239 base pairs of the G gene revealed that field strains and not vaccine strains were recovered from these flocks. Overall, the virological examination confirmed the high prevalence of aMPV in Israel and revealed a co-circulation of two subtypes, A and B, with aMPV subtype B being the dominant subtype.

Argasid ticks as possible vectors of West Nile virus in Israel.

Mites and soft ticks collected directly from wild and domestic birds and their nests were tested for the presence of West Nile virus (WNV). The cattle egret argas, Argas arboreus, was collected from the nests of seven cattle egret colonies. Out of 1,000 A. arboreus pools examined, 16 were positive for WNV based on RT-PCR technique. The positive pools were from four nesting colonies of birds. Out of 37 cattle egret squabs examined, 37.8% had serum-neutralizing antibodies to WNV. WNV RNA was also detected in one out of 15 pools of R. turanicus, in one out of 21 pools of O. sylviarum, and in one out of 18 pools of D. gallinae, while 63 pools of A. reflexus, 11 of R. sanguineus, and 30 of Hyalomma spec. were negative. The role of mites and ticks in maintaining the endemic state of WNV in Israel is discussed.

Comparison of serological and virological findings from subgroup J avian leukosis virus-infected neoplastic and non-neoplastic flocks in Israel.

Blood samples from nine broiler breeder flocks comprising five flocks clinically affected with myeloid leukosis tumours (ML+) and four tumour-free flocks from the same commercial background (ML-) were compared for avian leukosis virus subgroup J (ALV-J) serum antibodies by enzyme-linked immunosorbent assay (ELISA), for antigenemia (group-specific antigen) by antigen-trapping ELISA and for viremia. Group-specific antigen was detected in the sera of 58.1% of ML+ birds and 46.4% of the ML- birds (P=not significant), while 45.5% of ML+ birds and 24.1% of the ML- birds had ALV-J antibodies (P=0.065). In inoculated cell culture, 64.1% of the ML+ sera were viremic compared with 16.7% of the ML- sera (P=0.001). Similar significant differences were found between the two groups of flocks when ALV-J viremia was detected by immunofluorescence using a monoclonal env antibody (P=0.004), and for proviral DNA by polymerase chain reaction using two different sets of env-gene primers, H5-H7 (P=0.001) and R5-F5 (P=0.001). Using the primer pair R5-F5 the product size was approximately 1 kbp, while some heterogeneity in size among isolates was discernable. Our results indicate that a combination of diagnostic tests should be adopted in routine examination of tumour material in order to rule out false-negative findings.

Akabane virus in Israel: a new virus lineage.

This report describes the first molecular characterization of Akabane virus (AKAV) in Israel. The virus was recognized by real-time RT-PCR in extracts from Culicoides imicola insects trapped at the Volcani Center located in the center of Israel. This is also the first report on the use of real-time RT-PCR to identify the virus. The quantitative capability of this technique was applied, and it was calculated that the insect extract contains 1.5 x 10(5) copies of the genome segment S. Following amplification of the small (S) genome segment, its nucleotide sequence was determined to have 93.4% identity or greater with the S segment of other AKAV isolates. The deduced amino acid (aa) sequence of the combined nucleocapsid and the non-structural protein showed more than 96.6% identity. Phylogentic trees constructed using the combined deduced nucleocapsid and the non-structural protein aa sequences showed that the Israeli isolate forms a fourth cluster of AKAV, indicating a separate virus lineage. Attempts to isolate the virus by inoculation to Vero cells and by intracerebral inoculation to mice were unsuccessful.

A twelve-month study of West Nile virus antibodies in a resident and a migrant species of kestrels in Israel.

Two species of kestrel, the common and lesser, were caught each month at three geographically defined locations in Israel over a 12-month period, and a total of 306 blood samples were examined for West Nile virus neutralizing antibodies. The prevalences and mean antibody titers were analyzed statistically by the multiple linear regression model and were shown to be significantly affected by two of the independent variables, location and age of the bird. The season had no overall effect on prevalence and titer but a comparison of the mean monthly titers revealed that April was highest and July and August the lowest statistically for the common kestrel which is a resident species. In contrast, the migrating lesser kestrel was caught only in the spring months and principally at the Jerusalem location, where eight out of 29 birds were seropositive. By comparing the serology of the non-migrating, common kestrel with the migrating, lesser kestrel, the effect of seasonality was evaluated in relation to their ecological patterns and yielded evidence for the entry in April of a small number of previously infected common kestrels into Israel. This serological approach based on continuous sampling over an extended period could be used to forecast in the coming years the timing and dispersion of West Nile virus in both Old and New Worlds if surveys are based on a limited number of informative (flag) species.

Direct (non-vector) transmission of West Nile virus in geese.

During a recent epizootic, losses due to West Nile virus (WNV) infection in young goose flocks were estimated to be far greater than expected if mosquito-borne transmission was the principal route of infection. Contact transmission was investigated experimentally as an alternative explanation. A group of 10, 3-week-old geese were inoculated subcutaneously and placed in one insect-proof room with 20 geese of the same age. A group of 10 geese were housed in an adjacent insect-proof room to serve as an environmental control. All geese in the inoculated group produced antibodies, eight became viraemic and five died between 7 and 10 days after infection. Virus was shed from the cloaca and oral cavity by three geese. Two of the in-contact birds died on days 10 and 17 after infection, and WNV was recovered from another three birds. None of the environmental control group became infected. This result strongly suggests that horizontal transmission of WNV can occur in commercial flocks and may be aggravated if cannibalism and feather-picking of sick geese occur.

Phylogenetic relationships of West Nile viruses isolated from birds and horses in Israel from 1997 to 2001.

In November 1997, an outbreak of a neuroparalytic disease caused by West Nile (WN) virus was diagnosed in young goose flocks. Domestic geese were similarly affected in the late summer and fall of 1998, 1999, 2000 and 2001. WN viruses were also isolated from migratory and wild birds and horses in 1998-2001. A 1278 bp sequence of the envelope gene of 24 Israeli WN virus isolates was compared with those of seven isolates from Africa, Europe and New York. As a result, the Israeli isolates could then be grouped into two clusters. The 15 avian and three equine from 1997-2001 in the first cluster of viruses were shown to be identical to WN-NY99, while the second cluster comprised one goose isolate from 1998 and two goose and two pigeon isolates from 2000. These closely resembled the most recent Old World isolates, and indicate that at least two WN genotypes were co-circulating in the region during this time.