Ecology and molecular epidemiology of H9N2 avian influenza viruses isolated in Israel during 2000-2004 epizootic.

The first two isolates of H9N2 influenza virus were picked up from turkey and chicken hosts in May 2000, but the actual epizootic of the low pathogenicity avian influenza (LPAI) H9N2 virus started in December 2001, following a 1.5-year period of silence, during which the H10N7 and H6N3 influenza viruses were isolated sporadically. The outbreak of the H9N2 influenza began in northern Israel, from where the epizootic spread all over the country. Damage was relatively limited because of the widespread use of an inactivated vaccine. Single isolates were recorded in commercial ostrich and goose flocks, and in a wild pigeon. Apart from the routine serological tests, the diagnostics used the RT-PCR (reverse transcription polymerase chain reaction) test with type-specific primers related to the M and nucleoprotein (NP) genes, and a set of subtype-specific primers related to all the haemagglutinin (HA) and neuraminidase (NA) subtypes. All the primers were specially constructed. The part coding for N-terminus of the H chain of the HA gene of 61 out of 400 isolates was sequenced. The isolates showed a high rate of mutability, and differed distinctly from the H9 prototype strain; they belong to the same phylogenetic lineage divided into three sublineages, one of which exhibited a unique cleavage-site motif RSKR. The result indicates that two parallel evolutionary trends originated from the same local "prototype" isolate.

Nucleotide sequence of the matrix protein gene of avian paramyxovirus, serotype 3b: evidence on another member of the suggested new genus of the subfamily Paramyxovirinae.

The complete nucleotide sequence of the gene encoding the matrix protein (M) of the avian paramyxovirus, serotype 3b (APMV-3b), has been determined by means of the direct sequencing of viral RNA using reverse transcriptase reaction. The adjacent portions of the neighboring phosphoprotein (P) and fusion (F) protein genes were also sequenced that permitted to determine the consensus sequence of the viral genome, the poly(A) tract, downstream and upstream non-coding portions of the P and F genes, respectively, as well as the corresponding intergenic regions. The gene is 1478 nucleotides long with a protein-coding sequence of 1194 nucleotides. The deduced protein consists of 398 amino acids with a calculated MW 44,465. According to the multalignment and phylogenetic analyses, the APMV-3b M protein has shown the closest relatedness towards Newcastle disease virus (NDV) which has recently been suggested to be excluded from the Rubulavirus genus and assigned (together with APMV-6) to a new Avulavirus genus within the subfamily Paramyxovirinae of the Paramyxoviridae family. On the basis of the M protein genetic multalignment, phylogenetic relationships, bipartite nuclear localization signal identification in combination with the cysteine residues distribution, and by the degree of intrageneric heterogeneity, the APMV-3b is proposed to be another member (together with NDV and APMV-6) of the new genus.

Antigenic heterogeneity amongst the field isolates of Newcastle Disease Virus (NDV) in relation to the vaccine strain. Part II: studies on viruses isolated from domestic birds in Israel.

Forty three Newcastle disease virus (NDV) strains isolated before and during 1997 in Israel from domestic birds were studied by means of the three panels of monoclonal antibodies prepared against all the viral envelope proteins in order to reveal the possible antigenic differences between them and the VH strain used in Israel for poultry vaccination. Three isolates were found to have significant antigenic differences in the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins as compared to the vaccine strain. As to the matrix protein, almost all the viruses isolated during the year 1997 were found to have considerable differences from the vaccine strain in two of four antigenic sites.

Antigenic heterogeneity among the field isolates of Newcastle disease virus (NDV) in relation to the vaccine strain: 1. Studies on viruses isolated from wild birds in Israel.

In order to reveal the viruses strongly differing from the VH NDV strain used in Israel for poultry vaccination, 54 NDV strains isolated during the last 15 years in Israel from feral birds were studied by means of the panels of 39 monoclonal antibodies. Six isolates were found to have considerable antigenic differences in envelope proteins as compared to the vaccine strain. In four cases, the differences were related mostly to the hemagglutinin-neuraminidase glycoprotein, in one case to the fusion glycoprotein, and in one case to the matrix protein.

The comparative antigenic characterization of Newcastle disease virus strains isolated in Kenya and Kazakhstan.

Twenty six Newcastle disease viruses--12 reference strains and 14 strains isolated in Kenya and Kazakhstan--were characterized by means of a large panel of 38 monoclonal antibodies (MAB) directed against all the three envelope proteins: matrix, hemagglutinin-neuraminidase and fusion. The essential distinctions were revealed between the viruses isolated in Kenya and Kazakhstan while the differences amongst the viruses belonging to the same local group were much smaller. The heterogeneity amongst the viruses isolated in Kenya was more expressed as compared to the Kazakhstanian strains.

Antigenic characterization of the nucleocapsid protein of Newcastle disease virus by means of a new panel of monoclonal antibodies.

Nine monoclonal antibodies (MAB) against nucleocapsid protein (NP) of Newcastle disease virus (NDV) have been prepared and characterized. All the MABs were classified into three groups by means of the competitive binding assay. At least three antigenic sites were delineated on the NP. The 1st site includes two closely located epitopes; the 2nd site includes two related and two distinct epitopes; the 3rd site includes two closely related and one distinct epitopes.

Comparative characteristics of the Israeli Newcastle disease virus field strains by means of a wide panel of monoclonal antibodies against hemagglutinin-neuraminidase glycoprotein.

Fourteen mouse monoclonal antibodies (MAB) were tested for their ability to react with 15 reference and 52 local Newcastle disease virus (NDV) strains isolated in Israel during the last decade from feral birds. All the field isolates had no antigenic difference when examined by classic serological tests. However, MAB-mediated analysis revealed wide antigenic heterogeneity amongst the studied viruses. By the pattern of the MAB reactivity, all the isolates could be distributed into 13 groups.

Avian paramyxoviruses serotype 3 isolated from captive birds in Israel: clinical signs, pathology, and antigenic characterization.

Thirteen HA agents were isolated in Israel from captive flamingoes (Phoenicopterus ruber), Egyptian geese (Alopochen aegyptiacus) belonging to order Anseriformes, and ibis (Guara rubra) belonging to order Ciconiiformes. The isolation was done from postmortem materials in three cases of severe respiratory disease with high mortality. The isolates were examined serologically and identified as belonging to the serotype 3 of avian paramyxoviruses (APMV-3). The isolates were more close antigenically to the APMV-3b variety (prototype strain--APMV-3/Parakeet/Netherlands/449/75) than to the APMV-3a variety (prototype strain--APMV-3/Turkey/Wisconsin/68). This is the first isolation of APMV-3 from birds belonging to the orders Anseriformes and Ciconiiformes.

Variability of antigenic epitopes of the fusion protein of Newcastle disease virus.

Using a panel of 10 monoclonal antibodies (Mab) against fusion (F) protein of Newcastle disease virus (NDV), strain Australia-Victoria, three non-overlapping antigenic sites (F1, F2 and F3) and one site partially overlapping with the sites F1 and F2 (F1.2) have been identified. The sites F2 and F3 are clusters that each include four antigenic epitopes. The antigenic stability of the above epitopes was estimated by comparison of the binding capacity of the corresponding Mabs towards 63 NDV strains isolated in different years and places from various avian species. The results demonstrated high variability of the site F1.2 and of all the four epitopes of the site F2. At the same time, the only epitope of the site F1 can be defined as highly conservative: the corresponding Mab gave positive binding with 60 from 63 NDV strains, one from the four epitopes pertaining to site F3 was the most conservative--the corresponding Mab reacted with all the 63 strains used in the studies, while the other three Mabs showed rather low stability--the corresponding Mabs reacted with 34-39 NDV strains. Thus, as opposed to the published data asserting antigenic stability of the F protein contrary to the high variability of the haemagglutinin-neuraminidase (HN) protein, our results have revealed a number of variable epitopes on the F protein. This demonstrates an evolutionary changeability of the F protein, which is of importance from the theoretical (viral antigenic evolution) as well as practical point of view.

Isolation of avian serotype 3 paramyxoviruses from imported caged birds in Israel.

Ten avian serotype 3 paramyxoviruses were isolated for the first time in Israel from passerine and psittacine imported caged birds. The birds were submitted for investigation of an illness characterized by nonspecific signs of weakness, anorexia, vomiting, and sneezing. In addition, only the parakeets developed specific neurologic signs. In bacteriologic and pathologic investigation, cachexia and diarrhea were observed in both groups of birds. In psittacines, considerable alterations were observed in lungs, liver, and spleen. Some nonviral pathogens were occasionally isolated. The isolates appeared to belong to serotype 3b avian paramyxovirus (APMV), the prototype strain of which is APMV-3b/parakeet/Netherlands/449/75. The isolation of APMV-3 viruses from imported caged birds may represent a way of introduction of these viruses into the country.

Antigenic epitope characterization of matrix protein of Newcastle disease virus using monoclonal antibody approach: contrasting variability amongst NDV strains.

A panel of 15 monoclonal antibodies (MABs) against matrix (M) protein of Newcastle disease virus (NDV) was obtained and the specificity towards the M protein was proven by radioimmunoprecipitation assay and antigen capture enzyme-linked immunosorbent assay (ELISA). Further studies were directed to antigenic epitope mapping of the M protein by means of this panel. The epitope characterization was performed by competitive antibody-binding assay by means of labelling each MAB with biotin [3]. At least three clear non-overlapping and two partially overlapping groups were determined, each including four, one, eight, one, and one MAB, respectively. All the above MABs appeared to be induced by structural epitopes formed in conditions of tertiary structure of the native M antigen. Twelve reference and 51 recently isolated local NDV strains have been studied by means of this MAB panel, several lineages having been revealed. The high stability of some epitopes and different variability of the others was demonstrated. No correlation between the above lineages and some other properties of the studied NDV strains (host specificity, date and place of isolation) has been found.

Mixed paramyxovirus infection of wild and domestic birds in Israel.

Five cases of dual isolations of different serotypes of avian paramyxoviruses (APMV) from domestic and wild birds are described: one case of mixed infection by APMV-1 and APMV-4 and four cases of infection by APMV-1 and APMV-2 serotypes. The double infection was proven by consecutive isolations of two viruses from allantoic fluid samples derived from single swabs after their respective treatment by antisera against each suspected virus. The finding of double APMV infection in poultry farms appears to be important for epizootiology and pathogenesis of APMV-caused diseases.

Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity potential.

The deduced amino acid sequence at the hemagglutinin (HA) cleavage site of 76 avian influenza (AI) viruses, subtypes H5 and H7, was determined by reverse transcription-polymerase chain reaction and cycle sequencing techniques to assess pathogenicity. Eighteen of the 76 viruses were isolated in 1993 and 1994 from various sources in the United States. In addition, 34 H5 (4 highly pathogenic [HP] and 30 non-highly pathogenic [non-HP]) and 24 H7 (3 HP and 21 non-HP) repository viruses, isolated between 1927 and 1992, were sequenced and the sequences compared to those in recent isolates. All repository HP H5 and H7 viruses studied had multiple basic amino acids adjacent to the HA cleavage site and most had basic amino acids in excess of the proposed minimum motif B-X-B-R (B = basic amino acids arginine or lysine, X = nonbasic amino acid, R = arginine) that has been associated with high pathogenicity. Of the non-HP viruses studied, 35 of 38 for H5 and 30 of 31 for H7 conformed to the motif B-X-X-R and B-X-R, respectively. Two non-HP H5 viruses had the motif X-X-X-R at the cleavage site and a third had the motif B-X-X-K (K = basic amino acid lysine). One non-HP H7 (A/Pekin robin/CA/30412-5/94) had four basic amino acids (K-R-R-R) adjacent to the cleavage site. Although the Pekin robin isolate did not produce disease in chickens under the conditions of the study it did have the amino acid sequence compatible with that in HP AI viruses and, therefore, is considered potentially HP. This is the first account of an H7 virus that is non-HP in chickens but meets the molecular criterion to be classified as HP.

Antigenic heterogeneity of avian paramyxoviruses of serotype 2 ("Yucaipa-like") isolated from domestic and wild birds in Israel.

Thirty-three viruses of PMV-2 serotype isolated in Israel from domestic and wild birds during epizooty of a respiratory disease in 1979-1981 were studied comprehensively in comparison with a set of reference PMV-2 viruses using cross reaction hemagglutination inhibition tests, elution-hemagglutination pattern and pattern of migration of viral proteins in polyacrylamide gel electrophoresis. The results demonstrated considerable heterogeneity amongst the local isolates by the above three criteria which were not correlated with each other. There was no significant correlation between the differences found and chronology of isolation of the viruses. The results indicated that there may have been simultaneous co-circulation of different PMV-2 strains in the local avian reservoir at the beginning of the epizootic rather than temporal accumulation of variants as a result of antigenic drift of an "ancestor" virus which caused the initial infection.

Antigenic heterogeneity of N2 neuraminidases of avian influenza viruses isolated in Israel.

Twenty one N2 neuraminidase (NA)-containing viruses isolated in Israel from different avian hosts during 1971-1984 were studied comparatively by means of the panel of 7 monoclonal antibodies (MAB) against A/Guiyang/57(H2N2) virus. Fifteen from the 21 viruses were studied in comprehensive cross reaction NA inhibition (NI) tests with the corresponding polyclonal antisera. The principal result of the studies is that all the isolates can be distributed into two main groups. The 1st group includes the majority of the isolates whose NA shows close relatedness to the "early" (1957 type) N2 NA by NI tests with polyclonal antisera, and demonstrates remarkable stability in the NI tests by reacting with the same 6 from 7 MABs of the panel. The 2nd group does not show any special kinship to either "early" or "late" (1968 type) N2 when analyzed with polyclonal antisera and demonstrates heterogeneity by the analysis with the MABs. A hypothetical explanation of the phenomenon of co-circulation in the local avian reservoir of viral strains displaying either remarkable stability or wide heterogeneity of their NAs is suggested. In accordance with it, the viruses with "stable" ("conservative") N2 NA did not leave the avian reservoir and, hence, did not drift because of very low antibody "selection pressure". Contrary to it, the viruses with heterogeneous N2 NA had been circulating in the human (mammalian) reservoir during various periods before their transfer into the avian reservoir; they drifted accordingly and, being then isolated from birds and designated as "avian" viruses, demonstrate heterogeneity of their NAs which is typical for human viruses.